29 human secreted proteins

ABSTRACT

The present invention relates to novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to these novel human secreted proteins.

[0001] This application is a continuation-in-part of, and claims benefitunder 35 U.S.C. § 120 of copending U.S. patent application Ser. No.PCT/US98/17709 filed Aug. 27, 1998, which is hereby incorporated byreference, which claims benefit under 35 U.S.C. § 119(e) based on U.S.Provisional Applications: Filing Date Appln No. 1. 29-Aug-199760/056,270 2. 29-Aug-1997 60/056,271 3. 29-Aug-1997 60/056,247 4.29-Aug-1997 60/056,073

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides andthe polypeptides encoded by these polynucleotides, uses of suchpolynucleotides and polypeptides, and their production.

BACKGROUND OF THE INVENTION

[0003] Unlike bacterium, which exist as a single compartment surroundedby a membrane, human cells and other eucaryotes are subdivided bymembranes into many functionally distinct compartments. Eachmembrane-bounded compartment, or organelle, contains different proteinsessential for the function of the organelle. The cell uses “sortingsignals,” which are amino acid motifs located within the protein, totarget proteins to particular cellular organelles.

[0004] One type of sorting signal, called a signal sequence, a signalpeptide, or a leader sequence, directs a class of proteins to anorganelle called the endoplasmic reticulum (ER). The ER separates themembrane-bounded proteins from all other types of proteins. Oncelocalized to the ER, both groups of proteins can be further directed toanother organelle called the Golgi apparatus. Here, the Golgidistributes the proteins to vesicles, including secretory vesicles, thecell membrane, lysosomes, and the other organelles.

[0005] Proteins targeted to the ER by a signal sequence can be releasedinto the extracellular space as a secreted protein. For example,vesicles containing secreted proteins can fuse with the cell membraneand release their contents into the extracellular space—a process calledexocytosis. Exocytosis can occur constitutively or after receipt of atriggering signal. In the latter case, the proteins are stored insecretory vesicles (or secretory granules) until exocytosis istriggered. Similarly, proteins residing on the cell membrane can also besecreted into the extracellular space by proteolytic cleavage of a“linker” holding the protein to the membrane.

[0006] Despite the great progress made in recent years, only a smallnumber of genes encoding human secreted proteins have been identified.These secreted proteins include the commercially valuable human insulin,interferon, Factor VIII, human growth hormone, tissue plasminogenactivator, and erythropoeitin. Thus, in light of the pervasive role ofsecreted proteins in human physiology, a need exists for identifying andcharacterizing novel human secreted proteins and the genes that encodethem. This knowledge will allow one to detect, to treat, and to preventmedical disorders by using secreted proteins or the genes that encodethem.

SUMMARY OF THE INVENTION

[0007] The present invention relates to novel polynucleotides and theencoded polypeptides. Moreover, the present invention relates tovectors, host cells, antibodies, and recombinant methods for producingthe polypeptides and polynucleotides. Also provided are diagnosticmethods for detecting disorders related to the polypeptides, andtherapeutic methods for treating such disorders. The invention furtherrelates to screening methods for identifying binding partners of thepolypeptides.

DETAILED DESCRIPTION Definitions

[0008] The following definitions are provided to facilitateunderstanding of certain terms used throughout this specification.

[0009] In the present invention, “isolated” refers to material removedfrom its original environment (e.g., the natural environment if it isnaturally occurring), and thus is altered “by the hand of man” from itsnatural state. For example, an isolated polynucleotide could be part ofa vector or a composition of matter, or could be contained within acell, and still be “isolated” because that vector, composition ofmatter, or particular cell is not the original environment of thepolynucleotide.

[0010] In the present invention, a “secreted” protein refers to thoseproteins capable of being directed to the ER, secretory vesicles, or theextracellular space as a result of a signal sequence, as well as thoseproteins released into the extracellular space without necessarilycontaining a signal sequence. If the secreted protein is released intothe extracellular space, the secreted protein can undergo extracellularprocessing to produce a “mature” protein. Release into the extracellularspace can occur by many mechanisms, including exocytosis and proteolyticcleavage.

[0011] In specific embodiments, the polynucleotides of the invention areless than 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb inlength. In a further embodiment, polynucleotides of the inventioncomprise at least 15 contiguous nucleotides of the coding sequence, butdo not comprise all or a portion of any intron. In another embodiment,the nucleic acid comprising the coding sequence does not contain codingsequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene in thegenome).

[0012] As used herein, a “polynucleotide” refers to a molecule having anucleic acid sequence contained in SEQ ID NO:X or the cDNA containedwithin the clone deposited with the ATCC. For example, thepolynucleotide can contain the nucleotide sequence of the full lengthcDNA sequence, including the 5′ and 3′ untranslated sequences, thecoding region, with or without the signal sequence, the secreted proteincoding region, as well as fragments, epitopes, domains, and variants ofthe nucleic acid sequence. Moreover, as used herein, a “polypeptide”refers to a molecule having the translated amino acid sequence generatedfrom the polynucleotide as broadly defined.

[0013] In the present invention, the full length sequence identified asSEQ ID NO:X was often generated by overlapping sequences contained inmultiple clones (contig analysis). A representative clone containing allor most of the sequence for SEQ ID NO:X was deposited with the AmericanType Culture Collection (“ATCC”). As shown in Table 1, each clone isidentified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number.The ATCC is located at 10801 University Boulevard, Manassas, Va.20110-2209, USA. The ATCC deposit was made pursuant to the terms of theBudapest Treaty on the international recognition of the deposit ofmicroorganisms for purposes of patent procedure.

[0014] A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:X, the complementthereof, or the cDNA within the clone deposited with the ATCC.“Stringent hybridization conditions” refers to an overnight incubationat 42° C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

[0015] Also contemplated are nucleic acid molecules that hybridize tothe polynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37° C. in asolution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;followed by washes at 50° C. with 1×SSPE, 0.1% SDS. In addition, toachieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

[0016] Note that variations in the above conditions may be accomplishedthrough the inclusion and/or substitution of alternate blocking reagentsused to suppress background in hybridization experiments. Typicalblocking reagents include Denhardt's reagent, BLOTTO, heparin, denaturedsalmon sperm DNA, and commercially available proprietary formulations.The inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems withcompatibility.

[0017] Of course, a polynucleotide which hybridizes only to polyA+sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in thesequence listing), or to a complementary stretch of T (or U) residues,would not be included in the definition of “polynucleotide,” since sucha polynucleotide would hybridize to any nucleic acid molecule containinga poly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

[0018] The polynucleotide of the present invention can be composed ofany polyribonucleotide or polydeoxribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. For example,polynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, the polynucleotidecan be composed of triple-stranded regions comprising RNA or DNA or bothRNA and DNA. A polynucleotide may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

[0019] The polypeptide of the present invention can be composed of aminoacids joined to each other by peptide bonds or modified peptide bonds,i.e., peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational prccessing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,aridation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, pegylation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination. (See, for instance,PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990);Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

[0020] “SEQ ID NO:X” refers to a polynucleotide sequence while “SEQ IDNO:Y” refers to a polypeptide sequence, both sequences identified by aninteger specified in Table 1.

[0021] “A polypeptide having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide of the present invention (i.e., the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to thepolypeptide of the present invention.)

Polynucleotides and Polypeptides of the Invention FEATURES OF PROTEINENCODED BY GENE NO: 1

[0022] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: HVNGNKXMDHHXQVAAALELVDPPGCRNSARGN(SEQ ID NO:69). Polynucleotides encoding these polypeptides are alsoencompassed by the invention. The gene encoding the disclosed cDNA isbelieved to reside on chromosome 8. Accordingly, polynucleotides relatedto this invention are useful as a marker in linkage analysis forchromosome 8.

[0023] This gene is expressed primarily in anergic T-cells.

[0024] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immune orhematopoietic disorders, particularly inflammatory disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune or hematopoietic system,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cells (e.g., immune,hematopoietic, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

[0025] The tissue distribution in anergic T-cells indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Expression of this gene product indicates a role in the regulation ofthe proliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses).

[0026] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis. and tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0027] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:11 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 852 of SEQID NO:11, b is an integer of 15 to 866, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:11, and where bis greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 2

[0028] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: EISNYV (SEQ ID NO:70).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0029] This gene is expressed primarily in anergic T-cells.

[0030] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immune orhematopoietic diseases and disorders, particularly inflammatoryconditions. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune or hematopoietic system, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., immune, hematopoietic, and cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0031] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 41 as residues: Arg-26 to Gln-32.

[0032] The tissue distribution in anergic T-cells indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Expression of this gene product indicates a role in the regulation ofthe proliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses).

[0033] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis. and tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0034] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:12 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 637 of SEQID NO:12, b is an integer of 15 to 651, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:12, and where bis greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 3

[0035] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence:IRHESKSMFVYSPNLSNAKGWHRGQCQAVPGYYLPLRK NS (SEQ ID NO:71).Polynucleotides encoding these polypeptides are also encompassed by theinvention. The gene encoding the disclosed cDNA is believed to reside onchromosome 13. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 13.

[0036] This gene is expressed primarily in spinal cord.

[0037] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, neural orcentral nervous system diseases and disorders, particularlyneurodegenerative conditions. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe neural system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., neural, spinal cord, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0038] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 42 as residues: Lys-25 to Ser-33.

[0039] The tissue distribution in spinal cord indicates the proteinproduct of this gene is useful for the detection, treatment, and/orprevention of neurodegenerative disease states, behavioral disorders, orinflammatory conditions which include, but are not limited toAlzheimer's Disease, Parkinson's Disease, Huntington's Disease, TouretteSyndrome, meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischenua and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception.

[0040] In addition, elevated expression of this gene product in regionsof the brain indicates it plays a role in normal neural function.Potentially, this gene product is involved in synapse formation,neurotransmission, learning, cognition, homeostasis, or neuronaldifferentiation or survival. Protein, as well as, antibodies directedagainst the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0041] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:13 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1322 of SEQID NO:13, b is an integer of 15 to 1336, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:13, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 4

[0042] This gene is expressed primarily in fibrosarcoma.

[0043] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,musculoskeletal disorders, particularly proliferative disorders such asfibrosarcoma. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of themusculoskeletal system, expression of this gene at significantly higheror lower levels may be routinely detected in certain tissues or celltypes (e.g., musculoskeletal, proliferative, fibroids, and cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0044] The tissue distribution in fibrosarcoma indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the detection, treatment, and/or prevention of various muscledisorders, such as muscular dystrophy, cardiomyopathy, fibroids, myomas,and rhabdomyosarcomas. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0045] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:14 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 571 of SEQID NO:14, b is an integer of 15 to 585, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:14, and where bis greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 5

[0046] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: EKGEGRTPSFIPTASDPKGILGSLVTMDTT (SEQID NO:72). Polynucleotides encoding these polypeptides are alsoencompassed by the invention. The gene encoding the disclosed cDNA isbelieved to reside on chromosome 1. Accordingly, polynucleotides relatedto this invention are useful as a marker in linkage analysis forchromosome 1.

[0047] This gene is expressed primarily in fast growing tissues such ashuman whole embryo, fetal brain, and cancer tissues.

[0048] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,developmental or proliferative disorders, such as growth deficiencies ortumorgenesis. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thefast growing tissues such as fetal tissues and cancer tissues,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g.,proliferative, developmental, and cancerous and wounded tissues) orbodily fluids (e.g., amniotic fluid, lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0049] The tissue distribution within fetal tissue and other cellularsources marked by proliferating cells indicates this protein may play arole in the regulation of cellular division, and may show utility in thediagnosis and treatment of cancer and other proliferative disorders.Similarly, developmental tissues rely on decisions involving celldifferentiation and/or apoptosis in pattern formation. Dysregulation ofapoptosis can result in inappropriate suppression of cell death, asoccurs in the development of some cancers, or in failure to control theextent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA). Therefore, the polynucleotides and polypeptidesof the present invention are useful in treating, detecting, and/orpreventing said disorders and conditions, in addition to other types ofdegenerative conditions. Thus this protein may modulate apoptosis ortissue differentiation and is useful in the detection, treatment, and/orprevention of degenerative or proliferative conditions and diseases.

[0050] Alternatively, expression within human fetal brain indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the detection/treatment of neurodegenerative disease states andbehavioural disorders such as Alzheimers Disease, Parkinsons Disease,Huntingtons Disease, Tourette Syndrome, schizophrenia, mania, dementia,paranoia, obsessive compulsive disorder, panic disorder, learningdisabilities, ALS, psychoses, autism, and altered bahaviors, includingdisorders in feeding, sleep patterns, balance, and perception. Inaddition, the gene or gene product may also play a role in the treatmentand/or detection of developmental disorders associated with thedeveloping embryo, sexually-linked disorders, or disorders of thecardiovascular system. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0051] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:15 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1130 of SEQID NO:15, b is an integer of 15 to 1144, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:15, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 6

[0052] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: KLVSKGKVSE (SEQ ID NO:73).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0053] This gene is expressed primarily in stromal cells.

[0054] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immune orhematopoietic diseases and/or disorders, particularly defects of stromalcell development. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly ofstromal cells, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,immune, hematopoietic, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0055] The tissue distribution in stromal cells indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Expression of this gene product indicates a role in the regulation ofthe proliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses).

[0056] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis. and tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. The uses include bone marrow cellex- vivo culture, bone marrow transplantation, bone marrowreconstitution, radiotherapy or chemotherapy of neoplasia. Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

[0057] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:16 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1023 of SEQID NO:16, b is an integer of 15 to 1037, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:16, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 7

[0058] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: PLGKMRQLKGKPKKETSKDKKERKQA (SEQ IDNO:74). Polynucleotides encoding these polypeptides are also encompassedby the invention.

[0059] This gene is expressed primarily in in cancer tissues, as wellas, thyroid, and to a lesser extent in prostate cancer, testes, andmacrophage.

[0060] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,reproductive, endocrine, or immune disorders, particularly tumorgenesis,immuneodeficiencies, bacterial infections, or inflammatory disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the cancers andthyroid, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,reproductive, endocrine, immune, and cancerous and wounded tissues) orbodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0061] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 46as residues: Met-1 to Gln-7.

[0062] The tissue distribution in cancer tissues indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of cancer and other proliferativedisorders. Expression within tumor tissue and other cellular sourcesmarked by proliferating cells indicates this protein may play a role inthe regulation of cellular division, and may show utility in thediagnosis and treatment of cancer and other proliferative disorders.Similarly, developmental tissues rely on decisions involving celldifferentiation and/or apoptosis in pattern formation. Dysregulation ofapoptosis can result in inappropriate suppression of cell death, asoccurs in the development of some cancers, or in failure to control theextent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA). Therefore, the polynucleotides and polypeptidesof the present invention are useful in treating, detecting, and/orpreventing said disorders and conditions, in addition to other types ofdegenerative conditions. Thus this protein may modulate apoptosis ortissue differentiation and is useful in the detection, treatment, and/orprevention of degenerative or proliferative conditions and diseases.

[0063] Additionally, the expression in hematopoietic cells and tissuesindicates that this protein may play a role in the proliferation,differentiation, and/or survival of hematopoietic cell lineages. In suchan event, this gene may be useful in the treatment oflymphoproliferative disorders, and in the maintenance anddifferentiation of various hematopoietic lineages from earlyhematopoietic stem and committed progenitor cells. Similarly, embryonicdevelopment also involves decisions involving cell differentiationand/or apoptosis in pattern formation. Thus this protein may also beinvolved in apoptosis or tissue differentiation and could again beuseful in cancer therapy.

[0064] Alternatively, the tissue distribution within various endocrinetissues indicates that polynucleotides and polypeptides corresponding tothis gene are useful for the detection, treatment, and/or prevention ofvarious endocrine disorders and cancers, particularly Addison's disease,Cushing's Syndrome, and disorders and/or cancers of the pancrease (e.g.,diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g., hyper-,hypopituitarism), thyroid (e.g., hyper-, hypothyroidism), parathyroid(e.g., hyper-, hypoparathyroidism), hypothallamus, and testes.

[0065] Moreover, the expression within various reproductive organsindicates that the protein product of this gene may show utility in thedetection, treatment, and/or prevention of a variety of reproductivedisorders, particularly male infertility. Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

[0066] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:17 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 911 of SEQID NO:17, b is an integer of 15 to 925, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:17, and where bis greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 8

[0067] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: LLIAGTGSSLYWAFTV (SEQ ID NO:75).Polynucleotides encoding these polypeptides are also encompassed by theinvention. The gene encoding the disclosed cDNA is believed to reside onchromosome 12. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 12.

[0068] This gene is expressed primarily in brain, reproductive organs,and immune cell/tissues, and to a lesser extent, in other tissues.

[0069] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, neuronal,reproductive, or immune and inflammatory diseases. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the brain, reproductive organs, andimmune cell/tissues, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues of cell types(e.g., neural, reproductive, immune, and cancerous and wounded tissues)or bodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0070] The tissue distribution in brain indicates that polynucleotidesand polypeptides corresponding to this gene are useful for thedetection/treatment of neurodegenerative disease states and behaviouraldisorders such as Alzheimers Disease, Parkinsons Disease, HuntingtonsDisease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia,obsessive compulsive disorder, panic disorder, learning disabilities,ALS, psychoses , autism, and altered bahaviors, including disorders infeeding, sleep patterns, balance, and perception. In addition, the geneor gene product may also play a role in the treatment and/or detectionof developmental disorders associated with the developing embryo,sexually-linked disorders, or disorders of the cardiovascular system.

[0071] Alternatively, the secreted protein can also be used to determinebiological activity, to raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions and as nutritional supplements. It may also have a verywide range of biological acitivities. Typical of these are cytokine,cell proliferation/differentiation modulating activity or induction ofother cytokines; immunostimulating/immunosuppressant activities (e.g.,for treating human immunodeficiency virus infection, cancer, autoimmunediseases and allergy); regulation of hematopoiesis (e.g., for treatinganaemia or as adjunct to chemotherapy); stimulation or growth of bone,cartilage, tendons, ligaments and/or nerves (e.g., for treating wounds,stimulation of follicle stimulating hormone (for control of fertility);chemotactic and chemokinetic activities (e.g., for treating infections,tumors); hemostatic or thrombolytic activity (e.g., for treatinghaemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g.,for treating septic shock, Crohn's disease); as antimicrobials; fortreating psoriasis or other hyperproliferative diseases; for regulationof metabolism, and behaviour. Also contemplated is the use of thecorresponding nucleic acid in gene therapy procedures. Protein, as wellas, antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

[0072] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:18 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1183 of SEQID NO:18, b is an integer of 15 to 1197, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:18, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 9

[0073] This gene is expressed primarily in human tonsils and B-cellsfrom lymphoma patients.

[0074] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, disordersrelated to immune or hematopoietic system, particularly B-cell lymphoma.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the immune orhematopoietic system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., immune, hematopoietic, and cancerous and wounded tissues) orbodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0075] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 48 as residues: Ser-5 1 to Leu-60.

[0076] The tissue distribution in human tonsils and B-cells indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for the diagnosis and treatment of a variety of immune systemdisorders. Expression of this gene product indicates a role in theregulation of the proliferation; survival; differentiation; and/oractivation of potentially all hematopoietic cell lineages, includingblood stem cells. This gene product may be involved in the regulation ofcytokine production, antigen presentation, or other processes that mayalso suggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses).

[0077] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis inflammatory bowel disease, sepsis, acne,and psoriasis. and tissues. In addition, this gene product may havecommercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0078] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:19 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1092 of SEQID NO:19, b is an integer of 15 to 1106, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:19, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 10

[0079] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: GKRVXLQVPVRNSRVDPRVRVWGVVLNVCGPGAWGLAEHSVK (SEQ ID NO:76), KQCPALNGSFKGVGTNCKMMYLEXWGLRDILLPPRXFVADGSXQGGEREASVLFGKLAIKTGKGVLFSRKLDLFRTLSAPNRCGQLPAAQRDEGQRQDAAGKVNIVFVSTVEDKKGMKSTVRTIMVGE (SEQ ID NO:77),RDILLPPRXFVADGSXQGGEREASVLFG (SEQ ID NO:78), and/or LFRTLSAPNRCGQLPAAQRDEGQRQDAAGKVNWFVST (SEQ ID NO:79). Polynucleotides encodingthese polypeptides are also encompassed by the invention. The geneencoding the disclosed cDNA is believed to reside on chromosome 9.Accordingly, polynucleotides related to this invention are useful as amarker in linkage analysis for chromosome 9.

[0080] This gene is expressed primarily in resting T-cells, fetal liverand spleen.

[0081] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,developmental disorders, or disorders related to immune system,particularly immunodeficiencies and inflammatory disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., developmental, differentiating,immune, and cancerous and wounded tissues) or bodily fluids (e.g.,amniotic fluid, lymph, bile, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0082] The tissue distribution in resting T-cells, fetal liver andspleen indicates that polynucleotides and polypeptides corresponding tothis gene are useful for the diagnosis and treatment of a variety ofimmune or hematopoietic system disorders. Expression of this geneproduct indicates a role in the regulation of the proliferation;survival; differentiation; and/or activation of potentially allhematopoietic cell lineages, including blood stem cells. This geneproduct may be involved in the regulation of cytokine production,antigen presentation, or other processes that may also suggest ausefulness in the treatment of cancer (e.g., by boosting immuneresponses).

[0083] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis. and tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. The uses include bone marrow cellex- vivo culture, bone marrow transplantation, bone marrowreconstitution, radiotherapy or chemotherapy of neoplasia.

[0084] Alternatively, expression within fetal tissue and other cellularsources marked by proliferating cells indicates that this protein mayplay a role in the regulation of cellular division, and may show utilityin the diagnosis and treatment of cancer and other proliferativedisorders. Similarly, embryonic development also involves decisionsinvolving cell differentiation and/or apoptosis in pattern formation.Thus this protein may also be involved in apoptosis or tissuedifferentiation and could again be useful in cancer therapy. Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

[0085] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:20 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 918 of SEQID NO:20, b is an integer of 15 to 932, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:20, and where bis greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 11

[0086] The translation product of this gene shares sequence homologywith the human TATA-binding protein-associated factor dTAFII250 which isthought to be a general transcriptional activator of RNA polymerase II(See Genbank Accession no.R56493). In specific embodiments, polypeptidesof the invention comprise the following amino acid sequence:GRPTRPKTPYDESKFYIGCDLCTNWYHGECVGITEKEAKKMDVYICNDCKRAQEGSSEELYCICRTPYDESQFYIGCDRCQNWYHGRCVGILQSEAELIDEYVCPQCQSTEDAMTVLTPLTEKDYEGLKRVLRSLQAHKMAWPFLEPVDPNDAPDYYGVIKEPMDLATMEERVQRRYYEKLTEFVADMTKIFDNCRYY NPSDSPFYQCAEVLESFFVQKLKGFKASRSHNNKLQSTAS (SEQ ID NO:80), TPYDESKFYIGCDLCTNWYH (SEQID NO:81), EAKKMDVYICNDCKRAQE GSSEEL (SEQ ID NO:82),RCQNWYHGRCVGILQSEAELIDEYV (SEQ ID NO:83), STEDAMTVLTPLTEKDYEGLKR (SEQ IDNO:84), DPNDAPDYYGVIKEPMD LATM (SEQ ID NO:85), VDPRVRKRLM (SEQ IDNO:87), and/or EKLTEFVADMT KIFDNCRYYNP (SEQ ID NO:86). Polynucleotidesencoding these polypeptides are also encompassed by the invention.

[0087] This gene is expressed primarily in Jurkat T-cells and humanstriatum.

[0088] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, disordersof immune or nervous system. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe immune and nervous system, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., immune, hematopoietic, neural, and cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0089] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 50 as residues: Gln-24 to Lys-30, Gly-41 to Ala-46, Ala-97 toPro-103.

[0090] The tissue distribution in human striatum indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the detection/treatment of neurodegenerative disease states andbehavioural disorders such as Alzheimers Disease, Parkinsons Disease,Huntingtons Disease, Tourette Syndrome, schizophrenia, mania, dementia,paranoia, obsessive compulsive disorder, panic disorder, learningdisabilities, ALS, psychoses , autism, and altered bahaviors, includingdisorders in feeding, sleep patterns, balance, and preception. Inaddition, the gene or gene product may also play a role in the treatmentand/or detection of developmental disorders associated with thedeveloping embryo, sexually-linked disorders, or disorders of thecardiovascular system.

[0091] Alternatively, the tissue distribution indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Expression of this gene product indicates a role in the regulation ofthe proliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses).

[0092] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis. and tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0093] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:21 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1018 of SEQID NO:21, b is an integer of 15 to 1032, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:21, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 12

[0094] The gene encoding the disclosed cDNA is believed to reside onchromosome 14. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 14.

[0095] This gene is expressed primarily in human umbilical veinendothelial cells, breast, and to a lesser extent in placenta.

[0096] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, vascularor developmental disorders, particularly those involving the umbilicalcord or placenta, in addition to reproductive disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the endothelial system, expression ofthis gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., endothelial, vascular,reproductive, breast, developmental, and cancerous and wounded tissues)or bodily fluids (e.g., amniotic fluid, breast milk, serum, plasma,urine, synovial fluid and spinal fluid) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.

[0097] The tissue distribution in umbilical cord endothelial cellsindicates that polynucleotides and polypeptides corresponding to thisgene are useful for the diagnosis and treatment of cardiovasculardisorders, such as atherosclerosis, vasculitis, microvascular disease,vascular leak syndrome, embolism, stroke, aneurysm, and other relatedblood vessel disorders. Moreover, expression within embryonic tissue andother cellular sources marked by proliferating cells indicates that thisprotein may play a role in the regulation of cellular division, and mayshow utility in the diagnosis and treatment of cancer and otherproliferative disorders. Similarly, embryonic development also involvesdecisions involving cell differentiation and/or apoptosis in patternformation. Dysregulation of apoptosis can result in inappropriatesuppression of cell death, as occurs in the development of some cancers,or in failure to control the extent of cell death, as is believed tooccur in acquired immunodeficiency and certain neurodegenerativedisorders, such as spinal muscular atrophy (SMA).

[0098] Therefore, the polynucleotides and polypeptides of the presentinvention are useful in treating, detecting, and/or preventing saiddisorders and conditions, in addition to other types of degenerativeconditions. Thus this protein may modulate apoptosis or tissuedifferentiation and is useful in the detection, treatment, and/orprevention of degenerative or proliferative conditions and diseases, inaddition to cancer therapy. Protein, as well as, antibodies directedagainst the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0099] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:22 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 473 of SEQID NO:22, b is an integer of 15 to 487, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:22, and where bis greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 13

[0100] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence:CRELSSSCLSGRAPFKSSDCKERRSRCPRVPGFQNKNRV AILAELDKEKRKLL (SEQ ID NO:88).Polynucleotides encoding these polypeptides are also encompassed by theinvention. The gene encoding the disclosed cDNA is believed to reside onchromosome 9. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 9.

[0101] This gene is expressed primarily in human brain, fetal liverspleen, dendritic cells, and to a lesser extent in human B-celllymphoma.

[0102] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, neural,developmental, immune, or hematopoietic disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the nervous or immune system,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., neural,immune, hematopoietic, hepatic, developmental, reproductive, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, amnioticfluid, serum, plasma, urine, synovial fluid and spinal fluid) or anothertissue or cell sample taken from an individual having such a disorder,relative to the standard gene expression level, i.e., the expressionlevel in healthy tissue or bodily fluid from an individual not havingthe disorder.

[0103] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 52 as residues: Met-1 to Pro-10, Pro-19 to Gln-32, Gln-73 toAsp-80.

[0104] The tissue distribution in brain indicates that polynucleotidesand polypeptides corresponding to this gene are useful for thedetection/treatment of neurodegenerative disease states and behaviouraldisorders such as Alzheimers Disease, Parkinsons Disease, HuntingtonsDisease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia,obsessive compulsive disorder, panic disorder, learning disabilities,ALS, psychoses , autism, and altered bahaviors, including disorders infeeding, sleep patterns, balance, and perception. In addition, the geneor gene product may also play a role in the treatment and/or detectionof developmental disorders associated with the developing embryo,sexually-linked disorders, or disorders of the cardiovascular system.

[0105] Alternatively, the expression within various immune cells, inaddition to fetal liver/spleen indicates that polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosis andtreatment of a variety of immune system disorders. Expression of thisgene product in B-cell lymphoma and dendritic cells indicates a role inthe regulation of the proliferation; survival; differentiation; and/oractivation of potentially all hematopoietic cell lineages, includingblood stem cells. This gene product may be involved in the regulation ofcytokine production, antigen presentation, or other processes that mayalso suggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses).

[0106] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis. and tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types.

[0107] Moreover, expression within fetal tissue and other cellularsources marked by proliferating cells indicates this protein may play arole in the regulation of cellular division, and may show utility in thediagnosis and treatment of cancer and other proliferative disorders.Similarly, developmental tissues rely on decisions involving celldifferentiation and/or apoptosis in pattern formation. Dysregulation ofapoptosis can result in inappropriate suppression of cell death, asoccurs in the development of some cancers, or in failure to control theextent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA). Therefore, the polynucleotides and polypeptidesof the present invention are useful in treating, detecting, and/orpreventing said disorders and conditions, in addition to other types ofdegenerative conditions. Thus this protein may modulate apoptosis ortissue differentiation and is useful in the detection, treatment, and/orprevention of degenerative or proliferative conditions and diseases.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0108] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:23 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1446 of SEQID NO:23, b is an integer of 15 to 1460, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:23, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 14

[0109] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: IRHELKSWREENTSHPASGKGPLLD (SEQ IDNO:89), NLGGKKTHPTLPPGRGLSWTCLLQLLLSQMGTSRTDTSANSSSASLSSAPSCCVWESCSPSLGSRHANISPSQTAPWCSSGGAACAVVGLGGCDPGPLPGATSAPCKAAERSADGPRPSLHLWREPPVCPVPYLWVSVLDKRHAHQRPGHLGPWMWLQLGAGTAKRDRHWRLRAPDVWVPFSADHGALDCACGIVFLRGCPC (SEQ ID NO:90),TLPPGRGLSWTCLLQLLLSQM (SEQ ID NO:91), SANSSSASLSSAP SCCVWES (SEQ IDNO:92), PSQTAPWCSSGGAACAVVGLGGCDP (SEQ ID NO:93), APCKAAERSADGPRPSLHLWR(SEQ ID NO:94), QRPGHLGPWMW LQLGAGTAKRDRHWRLRA (SEQ ID NO:95),LRAPDVWVPFSADHGALDCAC GIVF (SEQ ID NO:96),APPRLPHGAQVAGLHVPWLGLGAVILARSRAQLQLRARLQRGQQMDPDRAFICGESRQFAQCLIFGFLFLTSGMLISVLGIWVPGCGSNWAQEPLNETDTGDSEPRMCGFLSLQIMGPLIVLVGLCFFVVAHVKKRNTLNAGQDASEREEGQIQIMEPVQVTVASAVAESPGTNSLLPNENPPSYYSIFNYGTPTSEGAASERDCESIYTISGTNSSSEASHTPHLPSELPPRYEEKENAAATFLPLSSE PSPP (SEQ IDNO:97), AQVAGLHVPWLGLGAVILARSRA (SEQ ID NO:98), RGQQMDPDRAFICGESRQFA(SEQ ID NO:99), LISVLGIWVPGCGSNWAQEP LNETDT (SEQ ID NO:100),TGDSEPRMCGFLSLQIMGPLIVLVGL (SEQ ID NO:101), DASEREEGQIQIMEPVQVTVASAVAE(SEQ ID NO:102), EREEG QIQINWPVQVTVASAVAESPGTNS (SEQ ID NO:103), and/orSEASHTPHLPS ELPPRYEEKENAAATFL (SEQ ID NO:104). Polynucleotides encodingthese polypeptides are also encompassed by the invention.

[0110] This gene is expressed primarily in human adult pulmonary tissue,and to a lesser extent in human kidney and messangial cell.

[0111] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,cardiopulmonary disorders, particularly respiratory system disorders,such as emphysema, or kidney disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the respiratory or renal system, expression of this geneat significantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., pulmonary, cardiovascular,endothelil, kidney, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, pulmonary surfactant, serum, plasma, urine, synovial fluidand spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0112] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 53 as residues: Glu-8 to Val-18.

[0113] The tissue distribution in pulmonary tissue indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of respiratory disorders,including emphysema, asthma, or bronchitis. Alternatively, expression inhuman kidney indicates that this gene or gene product could be used inthe treatment and/or detection of kidney diseases including renalfailure, nephritus, renal tubular acidosis, proteinuria, pyuria, edema,pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome,glomerulonephritis, hematuria, renal colic and kidney stones, inaddition to Wilms Tumor Disease, and congenital kidney abnormalitiessuch as horseshoe kidney, polycystic kidney, and Falconi's syndrome.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

[0114] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:24 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1084 of SEQID NO:24, b is an integer of 15 to 1098, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:24, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 15

[0115] This gene is expressed primarily in human cerebellum.

[0116] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, neural orcentral nervous system disorders, particularly neurodegenerativedisorders such as Alzheimers. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe nervous system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., neural, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

[0117] The tissue distribution in human cerebrum indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the detection/treatment of neurodegenerative disease states andbehavioural disorders such as Alzheimers Disease, Parkinsons Disease,Huntingtons Disease, Tourette Syndrome, schizophrenia, mania, dementia,paranoia, obsessive compulsive disorder, panic disorder, learningdisabilities, ALS, psychoses , autism, and altered bahaviors, includingdisorders in feeding, sleep patterns, balance, and perception. Inaddition, the gene or gene product may also play a role in the treatmentand/or detection of developmental disorders associated with thedeveloping embryo, sexually-linked disorders, or disorders of thecardiovascular system. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0118] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:25 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1001 of SEQID NO:25, b is an integer of 15 to 1015, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:25, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 16

[0119] When tested against K562 cell lines, supernatants removed fromcells containing this gene activated the ISRE (interferon- sensitiveresponsive element) promoter element. Thus, it is likely that this geneactivates leukmia cells, and to a lesser extent, immune or hematopoieticcells, through the Jaks-STAT signal transduction pathway. ISRE is apromoter element found upstream in many genes which are involved in theJaks-STAT pathway. The Jaks-STAT pathway is a large, signal transductionpathway involved in the differentiation and proliferation of cells.Therefore, activation of the Jaks-STATs pathway, reflected by thebinding of the ISRE element, can be used to indicate proteins involvedin the proliferation and differentiation of cells.

[0120] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence:GSRSRTFLSSSRPRVRPRVARRRQKGTAARRRQKGTAARRRQKGTAARRRQKGTAARRRQKGTALSPLRPSSSSLPQGXEAKPLHLFRAGXRPGXGNLVSESAGRSAGQGSPGPDA (SEQ ID NO:105), RQKGTAARRRQKGTAARRRQKGTAARRRQ (SEQ ID NO:106), and/or AKPLHLFRAGXRPGXG NLVSESAGR(SEQ ID NO:107). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0121] This gene is expressed primarily in human endothelial cells andhuman stromal endometrial fibroblasts.

[0122] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,integumentary or vascular diseases and/or disorders, particularlyendothelial or fibroblast related disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the integumentary system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., integumentary, endothelial, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

[0123] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 55 as residues: Glu-35 to Gln-40.

[0124] The tissue distribution in endothelial tissues and fibroblasts,combined with the observed ISRE biological activity indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment, diagnosis, and/or prevention of various skindisorders including congenital disorders (i.e. nevi, moles, freckles,Mongolian spots, hemangiomas, port-wine syndrome), integumentary tumors(i.e. keratoses, Bowen's disease, basal cell carcinoma, squamous cellcarcinoma, malignant melanoma, Paget's disease, mycosis fungoides, andKaposi's sarcoma), injuries and inflammation of the skin (i.e.wounds,rashes, prickly heat disorder, psoriasis, dermatitis), atherosclerosis,uticaria, eczema, photosensitivity, autoimmune disorders (i.e. lupuserythematosus, vitiligo, dermatomyositis, morphea, scleroderma,pemphigoid, and pemphigus), keloids, striae, erythema, petechiae,purpura, and xanthelasma.

[0125] Moreover, such disorders may predispose an individual (i.e.increased susceptibility) to viral and bacterial infections of the skin(i.e. cold sores, warts, chickenpox, molluscum contagiosum, herpeszoster, boils, cellulitis, erysipelas, impetigo, tinea, althletes foot,and ringworm). Moreover, the protein is useful in the detection,treatment, and/or prevention of a variety of vascular disorders andcondtions, which include, but are not limited to miscrovascular disease,vascular leak syndrome, aneurysm, stroke, embolism, thrombosis, coronaryartery disease, arteriosclerosis, and/or atherosclerosis. Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

[0126] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:26 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1183 of SEQID NO:26, b is an integer of 15 to 1197, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:26, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 17

[0127] The translation product of this gene shares sequence homologywith ATP/GTP binding site motif A of Saccharomyces cerevisiae (SeeGenbank Accession No.gnl|PID|e217937) which is thought to be importantin the regulation of various cellular processes, including transcriptionof important genes, in addition to signal transduction pathways.

[0128] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: TKFVQCPDGELQKRKEVVHTVSLHEIDVINSRTQGFLALFSGDTGEIKSEVREQINAKVAEWREEGKAEIIPGVLFIDEVHMLDESFSFLNRALESDMAPVLIMATNRGITRIRGTSYQSPHGIPIDLLDRLLIVSTTPYSEKDTKQILRIRCEEEDVEMSEDAYTVLTRIGLETSLRYAIQLITAASLVCRKRKGTEVQVDDIKRVYSLFLDESRSTQYMKEYQDAFLFNELKGETMDTS (SEQ ID NO:108),VVHTVSLHEIDVINSRTQGFLALF (SEQ ID NO:109), PGVLFIDEVH MLDIE (SEQ IDNO:110), or EEGKAEI (SEQ ID NO:111). Polynucleotides encoding thesepolypeptides are also encompassed by the invention. The gene encodingthe disclosed cDNA is believed to reside on chromosome 19. Accordingly,polynucleotides related to this invention are useful as a marker inlinkage analysis for chromosome 19.

[0129] This gene is expressed primarily in human adult testis, and to alesser extent, in activated T-cells.

[0130] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, malereproductive, endocrine, or immune diseases and/or disorders,particularly male infertility resulting from autoimmunity. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the male reproductive system,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., reproductive,endocrine, immune, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, seminal fluid, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0131] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 56 as residues: Arg-34 to His-42, Pro-59 to Gln-67, Arg-112to Thr-117, Glu-134 to Gln-139, Glu-156 to Ser-161.

[0132] The tissue distribution in human adult testis indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor diagnosis and treatment of a variety of male reproductive disorders,particularly male infertility. Similarly, the tissue distribution intestes indicates that polynucleotides and polypeptides corresponding tothis gene are useful for the detection, treatment, and/or prevention ofvarious endocrine disorders and cancers, particularly Addison's disease,Cushing's Syndrome, and disorders and/or cancers of the pancrease (e.g.,diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g., hyper-,hypopituitarism), thyroid (e.g., hyper-, hypothyroidism), parathyroid(e.g., hyper-,hypoparathyroidism) , hypothallamus, and testes.

[0133] Moreover, this gene product is also useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. Similarly, the protein is believed to be useful inthe treatment and/or diagnosis of testicular cancer. The testes are alsoa site of active gene expression of transcripts that may be expressed,particularly at low levels, in other tissues of the body. Therefore,this gene product may be expressed in other specific tissues or organswhere it may play related functional roles in other processes, such ashematopoiesis, inflammation, bone formation, and kidney function, toname a few possible target indications. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0134] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:27 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 988 of SEQID NO:27, b is an integer of 15 to 1002, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:27, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 18

[0135] The gene encoding the disclosed cDNA is believed to reside onchromosome 1. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 1. In specificembodiments, polypeptides of the invention comprise the following aminoacid sequence: LSQDLKGAR (SEQ ID NO:112), and/orLITHGCLSYYLLSLKLSSLLFFFFFLELLRIFPVWDPCTWFGFSLPCDNYNPDAS SFCLNYGSALP (SEQID NO:113). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0136] This gene is expressed primarily in human placental tissues.

[0137] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,reproductive or developmental disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly reproductive and developmental disorders, expression ofthis gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., reproductive,developmental, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0138] The tissue distribution in placental tissues indicates that theprotein product of this gene is useful for the diagnosis, treatment,and/or prevention of various reproductive, developmental, or congenitaldisorders, particularly Tay-Sachs disease, phenylkenonuria,galactosemia, hyperlipidemias, porphyrias, and Hurler's syndrome.

[0139] Alternatively, expression within placental tissue and othercellular sources marked by proliferating cells indicates that thisprotein may play a role in the regulation of cellular division, and mayshow utility in the diagnosis and treatment of cancer and otherproliferative disorders. Similarly, embryonic development also involvesdecisions involving cell differentiation and/or apoptosis in patternformation. Thus this protein may also be involved in apoptosis or tissuedifferentiation and could again be useful in cancer therapy. Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

[0140] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:28 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1313 of SEQID NO:28, b is an integer of 15 to 1327, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:28, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 19

[0141] The gene encoding the disclosed cDNA is believed to reside onchromosome 15. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 15. Inspecific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: IVLLFFFWMKTPAFPDSPPSSVLQFSEKSWDMWEGAWELGSLRLPGRQFRLCRKEQSPWEALGEGGAAAQH AWYCQPRGACV (SEQ IDNO:114). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0142] This gene is expressed primarily in human fetal brain tissue, andto a lesser extent in pineal gland, prostate, and thyroid tissues.

[0143] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,endocrine, reproductive, neural, or developmental disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells. particularly of the central nervous system, expressionof this gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., endocrine,reproductive, neural, developmental, and cancerous and wounded tissues)or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0144] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 58 as residues: Val-32 to Asn-40, Gly-59 to Gly-65.

[0145] The tissue distribution amongst various endocrine tissuesindicates that the protein product of this gene is useful for thedetection, treatment, and/or prevention of various endocrine disordersand cancers, particularly Addison's disease, Cushing's Syndrome, anddisorders and/or cancers of the pancrease (e.g., diabetes mellitus),adrenal cortex, ovaries, pituitary (e.g., hyper-, hypopituitarism),thyroid (e.g., hyper-, hypothyroidism), parathyroid (e.g., hyper-,hypoparathyroidism) , hypothallamus, and testes.

[0146] Alternatively, the tissue distribution in fetal brain tissueindicates that the protein product of this gene is useful for thedetection and/or treatment of neurodegenerative disease states andbehavioural disorders such as Alzheimers Disease, Parkinsons Disease,Huntingtons Disease, Tourette Syndrome, schizophrenia, mania, dementia,paranoia, obsessive compulsive disorder, panic disorder, learningdisabilities, ALS, psychoses, autism, and altered behaviors, includingdisorders in feeding, sleep patterns, balance, and perception. Inaddition, the gene or gene product may also play a role in the treatmentand/or detection of developmental disorders associated with thedeveloping embryo, sexually-linked disorders, or disorders of thecardiovascular system. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0147] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:29 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 654 of SEQID NO:29, b is an integer of 15 to 668, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:29, and where bis greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 20

[0148] This gene is expressed primarily in Merkel cells.

[0149] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,integumentary disorders or diseases. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune or integumentary system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., integumentary, immune, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

[0150] The tissue distribution in Merkel cells indicates that theprotein product of this gene is useful for the treatment, diagnosis,and/or prevention of various skin disorders including congenitaldisorders (i.e. nevi, moles, freckles, Mongolian spots, hemangiomas,port-wine syndrome), integumentary tumors (i.e. keratoses, Bowen'sdisease, basal cell carcinoma, squamous cell carcinoma, malignantmelanoma, Paget's disease, mycosis fungoides, and Kaposi's sarcoma),injuries and inflammation of the skin (i.e.wounds, rashes, prickly heatdisorder, psoriasis, dermatitis), atherosclerosis, uticaria, eczema,photosensitivity, autoimmune disorders (i.e. lupus erythematosus,vitiligo, dermatomyositis, morphea, scleroderma, pemphigoid, andpemphigus), keloids, striae, erythema, petechiae, purpura, andxanthelasma.

[0151] Moreover, such disorders may predispose increased susceptibilityto viral and bacterial infections of the skin (i.e. cold sores, warts,chickenpox, molluscum contagiosum, herpes zoster, boils, cellulitis,erysipelas, impetigo, tinea, althletes foot, and ringworm). Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

[0152] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:30 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1229 of SEQID NO:30, b is an integer of 15 to 1243, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:30, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 21

[0153] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence:MLJHTVIYLLDSISSNSFTTCVYLELFSIFLLFHSTICSEIESC (SEQ ID NO:115).Polynucleotides encoding, these polypeptides are also encompassed by theinvention.

[0154] This gene is expressed primarily in Merkel cells.

[0155] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,integumentary disorders, particularly in the diagnosis, treatment, orprevention of melanoma. Similarly, polypeptides and antibodies directedto these polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune and integumentary systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., integumentary, immune, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

[0156] The tissue distribution in Merkel cells indicates that theprotein product of this gene is useful for the treatment, diagnosis,and/or prevention of various skin disorders including congenitaldisorders (i.e. nevi, moles, freckles, Mongolian spots, hemangiomas,port-wine syndrome), integumentary tumors (i.e. keratoses, Bowen'sdisease, basal cell carcinoma, squamous cell carcinoma, malignantmelanoma, Paget's disease, mycosis fungoides, and Kaposi's sarcoma),injuries and inflammation of the skin (i.e.wounds, rashes, prickly heatdisorder, psoriasis, dermatitis), atherosclerosis, uticaria, eczema,photosensitivity, autoimmune disorders (i.e. lupus erythematosus,vitiligo, dermatomyositis, morphea, scleroderma, pemphigoid, andpemphigus), keloids, striae, erythema, petechiae, purpura, andxanthelasma. Moreover, such disorders may predispose increasedsusceptibility to viral and bacterial infections of the skin (i.e. coldsores, warts, chickenpox, molluscum contagiosum, herpes zoster, boils,cellulitis, erysipelas, impetigo, tinea, althletes foot, and ringworm).

[0157] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:31 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1039 of SEQID NO:31, b is an integer of 15 to 1053, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:31, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 22

[0158] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: KINNCFFKPHKKCIN (SEQ ID NO: 116).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0159] This gene is expressed primarily in Merkel cells.

[0160] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,integumentary or immune system diseases and disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune and integumentary systems,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., immune,integumentary, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

[0161] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 61 as residues: Ile-74 to His-79.

[0162] The tissue distribution in Merkel cells indicates that theprotein product of this gene is useful for the treatment, diagnosis,and/or prevention of various skin disorders including congenitaldisorders (i.e. nevi, moles, freckles, Mongolian spots, hemangiomas,port-wine syndrome), integumentary tumors (i.e. keratoses, Bowen'sdisease, basal cell carcinoma, squamous cell carcinoma, malignantmelanoma, Paget's disease, mycosis fungoides, and Kaposi's sarcoma),injuries and inflammation of the skin (i.e.wounds, rashes, prickly heatdisorder, psoriasis, dermatitis), atherosclerosis, uticaria, eczema,photosensitivity, autoimmune disorders (i.e. lupus erythematosus,vitiligo, dermatomyositis, morphea, scleroderma, pemphigoid, andpemphigus), keloids, striae, erythema, petechiae, purpura, andxanthelasma.

[0163] Moreover, such disorders may predispose increased susceptibilityto viral and bacterial infections of the skin (i.e. cold sores, warts,chickenpox, molluscum contagiosum, herpes zoster, boils, cellulitis,erysipelas, impetigo, tinea, althletes foot, and ringworm). Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

[0164] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:32 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1000 of SEQID NO:32, b is an integer of 15 to 1014, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:32, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 23

[0165] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: IRHEDASAVT (SEQ ID NO:117).Polynucleotides encoding these polypeptides are also encompassed by theinvention. The gene encoding the disclosed cDNA is thought to reside onchromosome 19. Accordingly, polynucleotides related to this inventionare useful as a marker in linkage analysis for chromosome 19.

[0166] This gene is expressed primarily in fetal liver spleen and invascular tissues such as fetal heart, embryonic and placental tissues,and to a lesser extent in, bone marrow stromal cells and Jurket membranebound polysomes.

[0167] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,developmental, vascular, or immune disorders, particularlyimmunodeficiencies or inflammatory disorders. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune and vascular systems, expression of this geneat significantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, hematopoietic, vascular,and cancerous and wounded tissues) or bodily fluids (e.g., lymph,amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid)or another tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

[0168] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 62 as residues: Leu-23 to Gln-29, Glu-33 to Ile-44.

[0169] The tissue distribution in fetal liver/spleen tissue indicatesthat the protein product of this gene is useful for the treatment and/ordiagnosis of hematopoietic related disorders such as anemia,pancytopenia, leukopenia, thrombocytopenia or leukemia since stromalcells are important in the production of cells of hematopoieticlineages. The uses include bone marrow cell ex vivo culture, bone marrowtransplantation, bone marrow reconstitution, radiotherapy orchemotherapy of neoplasia. The gene product may also be involved inlymphopoiesis, therefore, it can be used in immune disorders such asinfection, inflammation, allergy, immunodeficiency etc. In addition,this gene product may have commercial utility in the expansion of stemcells and committed progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types.

[0170] Similarly, expression within fetal tissues and other cellularsources marked by proliferating cells indicates that this protein mayplay a role in the regulation of cellular division, and may show utilityin the diagnosis and treatment of cancer and other proliferativedisorders. Similarly, embryonic development also involves decisionsinvolving cell differentiation and/or apoptosis in pattern formation.Thus this protein may also be involved in apoptosis or tissuedifferentiation and could again be useful in cancer therapy.Furthermore, the tissue distribution indicates that the protein productof this gene is useful for the diagnosis and/or treatment of disordersof the placenta. Specific expression within the placenta indicates thatthis gene product may play a role in the proper establishment andmaintenance of placental function.

[0171] Alternatively, this gene product may be produced by the placentaand then transported to the embryo, where it may play a crucial role inthe development and/or survival of the developing embryo or fetus.Expression of this gene product in a vascular-rich tissue such as theplacenta also indicates that, this gene product may be produced moregenerally in endothelial cells or within the circulation. In suchinstances, it may play more generalized roles in vascular function, suchas in angiogenesis. It may also be produced in the vasculature and haveeffects on other cells within the circulation, such as hematopoieticcells. It may serve to promote the proliferation, survival, activation,and/or differentiation of hematopoietic cells, as well as other cellsthroughout the body.

[0172] The tissue distribution in fetal heart tissue indicates that theprotein product of this gene is useful for the diagnosis and treatmentof conditions and pathologies of the cardiovascular system, such asheart disease, restenosis, atherosclerosis, stoke, angina, thrombosis,and wound healing. Protein, as well as, antibodies directed against theprotein may show utility as a tumor marker and/or immunotherapy targetsfor the above listed tissues.

[0173] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:33 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1359 of SEQID NO:33, b is an integer of 15 to 1373, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:33, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 24

[0174] The translation product of this gene shares sequence homologywith cni gene product (Comichon protein) of Drosophila melanogasterwhich is involved in signal processing during development (See GenbankAccession No.gil2460430).

[0175] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: SRPVMSGPGLYDPTTIMNADILAYCQKEGW (SEQID NO:118), FFCV MFLCAAEWLTLGLNMPLLAYHIWRYM (SEQ ID NO:119),WHIIAFDELKTDYKNPIDQCNTLNPLVLPEYLIHA (SEQ ID NO:120), GSPGCRNSATAXAPRSSSPA (SEQ ID NO:121), MAFTFAAFCYM (SEQ ID NO:122), and/orGMIYVLVSS (SEQ ID NO:123). Polynucleotides encoding these polypeptidesare also encompassed by the invention.

[0176] This gene is expressed primarily in melanocytes.

[0177] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,integumentary disorders, particularly melanocytoma and other cancers.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the immune andintegumentary systems, expression of this gene at significantly higheror lower levels may be routinely detected in certain tissues or celltypes (e.g., immune, integumentary, and cancerous and wounded tissues)or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0178] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 63 as residues: Leu-34 to Gln-44.

[0179] The tissue distribution in melanocytes indicates that the proteinproduct of this gene is useful for the treatment, diagnosis, and/orprevention of various skin disorders including congenital disorders(i.e. nevi, moles, freckles, Mongolian spots, hemangiomas, port-winesyndrome), integumentary tumors (i.e. keratoses, Bowen's disease, basalcell carcinoma, squamous cell carcinoma, malignant melanoma, Paget'sdisease, mycosis fungoides, and Kaposi's sarcoma), injuries andinflammation of the skin (i.e.wounds, rashes, prickly heat disorder,psoriasis, dermatitis), atherosclerosis, uticaria, eczema,photosensitivity, autoimmune disorders (i.e. lupus erythematosus,vitiligo, dermatomyositis, morphea, scleroderma, pemphigoid, andpemphigus), keloids, striae, erythema, petechiae, purpura, andxanthelasma.

[0180] Moreover, such disorders may predispose increased susceptibilityto viral and bacterial infections of the skin (i.e. cold sores, warts,chickenpox, molluscum contagiosum, herpes zoster, boils, cellulitis,erysipelas, impetigo, tinea, althletes foot, and ringworm). Based uponthe homology to the Cornichon protein, it may be inferred that theprotein product of this gene is useful for the detection, treatment,and/or prevention of a variety of proliferative disorders, particularlycancers, ulcers, or tumors. Protein, as well as, antibodies directedagainst the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0181] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:34 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1390 of SEQID NO:34, b is an integer of 15 to 1404, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:34, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 25

[0182] When tested against K562 cell lines, supernatants removed fromcells containing this gene activated the ISRE (interferon-sensitiveresponsive element) promoter element. Thus, it is likely that this geneactivates leukemia cells, and to a lesser extent other cells, throughthe Jak-STAT signal transduction pathway. ISRE is a promoter elementfound upstream in many genes which are involved in the Jaks-STATpathway. The Jaks-STAT pathway is a large, signal transduction pathwayinvolved in the differentiation and proliferation of cells. Therefore,activation of the Jaks-STATs pathway, reflected by the binding of theISRE element, can be used to indicate proteins involved in theproliferation and differentiation of cells.

[0183] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: LPTSRQSCQITYMATSLPQLLGSSKLSFMFNCFS(SEQ ID NO:124). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0184] This gene is expressed primarily in fetal liver/spleen tissue,and to a lesser extent, in human B cell lymphoma.

[0185] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to,developmental or immune disorders, particularly B cell diseases, such aslymphomas. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,developmental, immune, hematopoietic, and cancerous and wounded tissues)or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

[0186] The tissue distribution in fetal liver/spleen tissue and B celllymphoma indicates that the protein product of this gene is useful forthe diagnosis and/or treatment of a variety of immune system disorders.Expression of this gene product in fetal liver/spleen tissue indicates arole in the regulation of the proliferation; survival; differentiation;and/or activation of potentially all hematopoietic cell lineages,including blood stem cells. This gene product may be involved in theregulation of cytokine production, antigen presentation, or otherprocesses that may also suggest a usefulness in the treatment of cancer(e.g., by boosting immune responses).

[0187] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis. and tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types.

[0188] Similarly, expression within fetal tissue and other cellularsources marked by proliferating cells, combined with the detected ISREbiological activity, indicates that this protein may play a role in theregulation of cellular division, and may show utility in the diagnosisand treatment of cancer and other proliferative disorders. Similarly,embryonic development also involves decisions involving celldifferentiation and/or apoptosis in pattern formation. Thus this proteinmay also be involved in apoptosis or tissue differentiation and couldagain be useful in cancer therapy. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0189] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:35 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1079 of SEQID NO:35, b is an integer of 15 to 1093, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:35, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 26

[0190] The translation product of this gene shares sequence homologywith human complement decay-accelerating factor (DAF) (See GenbankAccession No. gil1857867). DAF is believed to play an important role inthe regulation of the complement cascade, specifically this proteinrecognizes c4b and c3b fragments that condense with cell-surfacehydroxyl or amino groups when nascent c4b and c3b are locally generatedduring c4 and c3 activation. interaction of daf with cell-associated c4band c3b polypeptides interferes with their ability to catalyze theconversion of c2 and factor b to enzymatically active c2a and bb andthereby prevents the formation of c4b2a and c3bbb, the amplificationconvertases of the complement cascade. In addition, mutation of DAF hasbeen found to lead to the Cromer blood group system which consists of atleast seven high-incidence (cr(a), tc(a), dr(a), es(a), wes(b), umc, andifc) and low-incidence (tc(b), tc(c), and wes(a)) antigens that resideon DAF. In the cromer phenotypes dr(a−) and inab there is reduced orabsent expression of DAF, respectively.

[0191] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence:NSFFFPCCFRYKLFGSTSSFCLISGSSVQWSDPXPECRGKS (SEQ ID NO:125).Polynucleotides encoding these polypeptides are also encompassed by theinvention. The gene encoding the disclosed cDNA is believed to reside onchromosome 1. Accordingly, polynucleotides related to this invention areuseful as a marker in linkage analysis for chromosome 1.

[0192] This gene is expressed primarily in activated human neutrophils.

[0193] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immune orhematopoietic disorders, particularly neutropenia, in addition todisorders related to the complement or neural system. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the hematopoietic, complement, orneural system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,immune, hematopoietic, neural and cancerous and wounded tissues) orbodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0194] The tissue distribution in neutrophils indicates that the proteinproduct of this gene is useful for the diagnosis and/or treatment of avariety of immune system disorders. Expression of this gene product inneutrophils indicates a role in the regulation of the proliferation;survival; differentiation; and/or activation of potentially allhematopoietic cell lineages, including blood stem cells. This geneproduct may be involved in the regulation of cytokine production,antigen presentation, or other processes that may also suggest ausefulness in the treatment of cancer (e.g., by boosting immuneresponses).

[0195] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis, and tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types.

[0196] Similarly, the tissue distribution in neutrophils, combined withthe homology to the DAF protein, indicates that the protein product ofthis gene is useful for the detection, treatment, and/or prevention of avariety of blood disorders, particularly complement disorders, ordisorders related to blood group antigens, such as the Cromer bloodgroup system (i.e. transfusions,etc.) Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0197] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:36 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 404 of SEQID NO:36, b is an integer of 15 to 418, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:36, and where bis greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 27

[0198] When tested against K562 cell lines, supernatants removed fromcells containing this gene activated the ISRE (interferon- sensitiveresponsive element) pathway. Thus, it is likely that this gene activatesleukemia cells, and to a lesser extent in other cells, through theJaks-STAT signal transduction pathway. ISRE is a promoter element foundupstream in many genes which are involved in the Jaks-STAT pathway. TheJaks-STAT pathway is a large, signal transduction pathway involved inthe differentiation and proliferation of cells. Therefore, activation ofthe Jaks-STATs pathway, reflected by the binding of the ISRE element,can be used to indicate proteins involved in the proliferation anddifferentiation of cells.

[0199] This gene is expressed primarily in neutrophils.

[0200] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immunedisorders, particularly neutropenia. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune or hematopoietic system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., immune, hematopoietic, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

[0201] Preferred epitopes include those comprising a sequence shown inSEQ ID NO. 66 as residues: Asp-45 to Thr-50.

[0202] The tissue distribution in neutrophils, combined with thedetected ISRE biological activity, indicates that the protein product ofthis gene is useful for the diagnosis and/or treatment of a variety ofimmune system disorders. Expression of this gene product in neutrophilsindicates a role in the regulation of the proliferation; survival;differentiation; and/or activation of potentially all hematopoietic celllineages, including blood stem cells. This gene product may be involvedin the regulation of cytokine production, antigen presentation, or otherprocesses that may also suggest a usefulness in the treatment of cancer(e.g., by boosting immune responses).

[0203] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis. and tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0204] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:37 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1028 of SEQID NO:37, b is an integer of 15 to 1042, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:37, and whereb is greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 28

[0205] When tested against K-562 cell lines, supernatants removed fromcells containing this gene activated the ISRE (interferon- sensitiveresponsive element) promoter element. Thus, it is likely that this geneactivates leukemia cells, and to a lesser extent, immune orhematopoietic cells, through the Jaks-STAT signal transduction pathwayISRE is a promoter element found upstream in many genes which areinvolved in the Jaks-STAT pathway. The Jaks-STAT pathway is a large,signal transduction pathway involved in the differentiation andproliferation of cells. Therefore, activation of the Jaks-STATs pathway,reflected by the binding of the ISRE element, can be used to indicateproteins involved in the proliferation and differentiation of cells.

[0206] This gene is expressed primarily in activated T-cells.

[0207] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immune orhematopoietic disorders, particularly immunodeficencies such as AIDs, orinflammatory disorders. Similarly, polypeptides and antibodies directedto these polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,immune, hematopoietic, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

[0208] The tissue distribution in activated T-cells indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Expression of this gene product in indicates a role in the regulation ofthe proliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses).

[0209] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis. and tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0210] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:38 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 784 of SEQID NO:38, b is an integer of 15 to 798, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:38, and where bis greater than or equal to a+14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 29

[0211] In specific embodiments, polypeptides of the invention comprisethe following amino acid sequence: VWVSGPWCTEFICLAVFTVTLQI (SEQ IDNO:126), TGG TAFLLMPNLLHLLLSPRRYPSVCRVSLGEWAMVHRVYLLGCFHCYSPNNDSVACLHIPSCCGIPPVLHSTAELCHS (SEQ ID NO:127), and/or RYPSVCRVSLGEWAMVHRVYLLGCFHCYSPNND (SEQ ID NO:128). Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

[0212] This gene is expressed primarily in neutrophils.

[0213] Therefore, polynucleotides and polypeptides of the invention areuseful as reagents for differential identification of the tissue(s) orcell type(s) present in a biological sample and for diagnosis ofdiseases and conditions which include, but are not limited to, immune orhematopoietic disorders, particularly neutropenia. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune or hemopoietic system,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., immune,hematopoietic, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

[0214] The tissue distribution in neutrophils indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and treatment of a variety of immune system disorders.Expression of this gene product indicates a role in the regulation ofthe proliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses).

[0215] Moreover, since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immune deficiency diseases such as AIDS,leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,acne, and psoriasis. and tissues. In addition, this gene product mayhave commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

[0216] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:39 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1016 of SEQID NO:39, b is an integer of 15 to 1030, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:39, and whereb is greater than or equal to a+14. 5′ NT NT of AA First Last ATCC SEQ5′ NT 3′ NT 5′ NT First SEQ AA AA First AA Last Deposit ID Total of ofof AA of ID of of of AA Gene cDNA Nr and NO: NT Clone Clone Start SignalNO: Sig Sig Secreted of No. Clone ID Date Vector X Seq. Seq. Seq. CodonPep Y Pep Pep Portion ORF  1 HSAXS66 209197 Uni-ZAP XR 11 866 1 866 102102 40 1 24 25 36 08/08/97  2 HSAYL90 209197 Uni-ZAP XR 12 651 1 651 188188 41 1 21 22 32 08/08/97  3 HSDIT06 209197 Uni-ZAP XR 13 1336 1 1336123 123 42 1 18 19 91 08/08/97  4 HSFAL43 209197 Uni-ZAP XR 14 585 1 585123 123 43 1 30 31 32 08/08/97  5 HSKEI54 209197 Uni-ZAP XR 15 1144 1751144 316 316 44 1 36 37 52 08/08/97  6 HSQBE28 209197 Uni-ZAP XR 16 1037229 1037 317 317 45 1 17 18 25 08/08/97  7 HTEAN76 209197 Uni-ZAP XR 17925 156 925 260 260 46 1 31 32 37 08/08/97  8 HTNAG39 209197 pBluescript18 1197 403 1191 468 468 47 1 45 46 70 08/08/97 SK-  9 HTODL90 209197Uni-ZAP XR 19 1106 1 1106 925 925 48 1 39 40 60 08/08/97 10 HTWDC20209197 pSport1 20 932 1 932 156 156 49 1 26 27 39 08/08/97 11 HUFAT34209197 pSport1 21 1032 1 1032 32 32 50 1 22 23 103 08/08/97 12 HUSGX69209197 pSport1 22 487 1 487 243 243 51 1 16 17 22 08/08/97 13 HAICJ23209197 Uni-ZAP XR 23 1460 1 1460 189 189 52 1 47 48 85 08/08/97 14HAPOF67 209197 Uni-ZAP XR 24 1098 1 1098 78 78 53 1 47 48 73 08/08/97 15HCEBR71 209197 Uni-ZAP XR 25 1015 1 1015 102 102 54 1 17 18 35 08/08/9716 HE8DG53 209197 Uni-ZAP XR 26 1197 307 1197 350 350 55 1 20 21 6708/08/97 17 HFSAY85 209197 Uni-ZAP XR 27 1002 457 1002 295 295 56 1 5859 161 08/08/97 18 HHEDD41 209197 pCMVSport 28 1327 154 1327 442 442 571 22 23 TF 08/08/97 3.0 19 HKCSO46 209197 pBluescript 29 668 20 668 217217 58 1 37 38 73 08/08/97 20 HKGAV60 209197 pSport1 30 1243 1 1243 192192 59 1 20 21 39 08/08/97 21 HKGDJ35 209197 pSport1 31 1053 1 1053 238238 60 1 19 20 32 08/08/97 22 HKGDJ66 209197 pSport1 32 1014 1 1014 215215 61 1 24 25 110 08/08/97 23 HMCDK27 209197 Uni-ZAP XR 33 1373 1 96933 33 62 1 17 18 75 08/08/97 24 HMCDX48 209197 Uni-ZAP XR 34 1404 1 140363 63 63 1 30 31 144 08/08/97 25 HMIAS24 209197 Uni-ZAP XR 35 1093 11093 178 178 64 1 42 43 44 08/08/97 26 HNFEG11 209197 Uni-ZAP XR 36 4181 418 251 251 65 1 31 32 47 08/08/97 27 HNGEP09 209197 Uni-ZAP XR 371042 1 1042 72 72 66 1 15 16 82 08/08/97 28 HTXKK52 209197 Uni-ZAP XR 38798 1 798 81 81 67 1 30 31 48 08/08/97 29 HNGJP90 209197 Uni-ZAP XR 391030 1 1030 157 157 68 1 14 15 94 08/08/97

[0217] Table 1 summarizes the information corresponding to each “GeneNo.” described above. The nucleotide sequence identified as “NT SEQ IDNO:X” was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table 1 and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually three to five overlapping sequences at each nucleotideposition), resulting in a final sequence identified as SEQ ID NO:X.

[0218] The cDNA Clone ID was deposited on the date and given thecorresponding deposit number listed in “ATCC Deposit No:Z and Date.”Some of the deposits contain multiple different clones corresponding tothe same gene. “Vector” refers to the type of vector contained in thecDNA Clone ID.

[0219] “Total NT Seq.” refers to the total number of nucleotides in thecontig identified by “Gene No.” The deposited clone may contain all ormost of these sequences, reflected by the nucleotide position indicatedas “5′ NT of Clone Seq.” and the “3′ NT of Clone Seq.” of SEQ ID NO:X.The nucleotide position of SEQ ID NO:X of the putative start codon(methionine) is identified as “5′ NT of Start Codon.” Similarly, thenucleotide position of SEQ ID NO:X of the predicted signal sequence isidentified as “5′ NT of First AA of Signal Pep.”

[0220] The translated amino acid sequence, beginning with themethionine, is identified as “AA SEQ ID NO:Y,” although other readingframes can also be easily translated using known molecular biologytechniques. The polypeptides produced by these alternative open readingframes are specifically contemplated by the present invention.

[0221] The first and last amino acid position of SEQ ID NO:Y of thepredicted signal peptide is identified as “First AA of Sig Pep” and“Last AA of Sig Pep.” The predicted first amino acid position of SEQ IDNO:Y of the secreted portion is identified as “Predicted First AA ofSecreted Portion.” Finally, the amino acid position of SEQ ID NO:Y ofthe last amino acid in the open reading frame is identified as “Last AAof ORF.”

[0222] SEQ ID NO:X and the translated SEQ ID NO:Y are sufficientlyaccurate and otherwise suitable for a variety of uses well known in theart and described further below. For instance, SEQ ID NO:X is useful fordesigning nucleic acid hybridization probes that will detect nucleicacid sequences contained in SEQ ID NO:X or the cDNA contained in thedeposited clone. These probes will also hybridize to nucleic acidmolecules in biological samples, thereby enabling a variety of forensicand diagnostic methods of the invention. Similarly, polypeptidesidentified from SEQ ID NO:Y may be used to generate antibodies whichbind specifically to the secreted proteins encoded by the cDNA clonesidentified in Table 1.

[0223] Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open readirig frameof over 1000 bases).

[0224] Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:X and the predicted translated amino acid sequence identified as SEQID NO:Y, but also a sample of plasmid DNA containing a human cDNA of theinvention deposited with the ATCC, as set forth in Table 1. Thenucleotide sequence of each deposited clone can readily be determined bysequencing the deposited clone in accordance with known methods. Thepredicted amino acid sequence can then be verified from such deposits.Moreover, the amino acid sequence of the protein encoded by a particularclone can also be directly determined by peptide sequencing or byexpressing the protein in a suitable host cell containing the depositedhuman cDNA, collecting the protein, and determining its sequence.

[0225] The present invention also relates to the genes corresponding toSEQ ID NO:X, SEQ ID NO:Y, or the deposited clone. The corresponding genecan be isolated in accordance with known methods using the sequenceinformation disclosed herein. Such methods include preparing probes orprimers from the disclosed sequence and identifying or amplifying thecorresponding gene from appropriate sources of genomic material.

[0226] Also provided in the present invention are species homologs.Species homologs may be isolated and identified by making suitableprobes or primers from the sequences provided herein and screening asuitable nucleic acid source for the desired homologue.

[0227] The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0228] The polypeptides may be in the form of the secreted protein,including the mature form, or may be a part of a larger protein, such asa fusion protein (see below). It is often advantageous to include anadditional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification, such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production.

[0229] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, including the secretedpolypeptide, can be substantially purified by the one-step methoddescribed in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides ofthe invention also can be purified from natural or recombinant sourcesusing antibodies of the invention raised against the secreted protein inmethods which are well known in the art.

Signal Sequences

[0230] Methods for predicting whether a protein has a signal sequence,as well as the cleavage point for that sequence, are available. Forinstance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses theinformation from a short N-terminal charged region and a subsequentuncharged region of the complete (uncleaved) protein. The method of vonHeinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information fromthe residues surrounding the cleavage site, typically residues −13 to+2, where +1 indicates the amino terminus of the secreted protein. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. (vonHeinje, supra.) However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

[0231] In the present case, the deduced amino acid sequence of thesecreted polypeptide was analyzed by a computer program called SignalP(Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), whichpredicts the cellular location of a protein based on the amino acidsequence. As part of this computational prediction of localization, themethods of McGeoch and von Heinje are incorporated. The analysis of theamino acid sequences of the secreted proteins described herein by thisprogram provided the results shown in Table 1.

[0232] As one of ordinary skill would appreciate, however, cleavagesites sometimes vary from organism to organism and cannot be predictedwith absolute certainty. Accordingly, the present invention providessecreted polypeptides having a sequence shown in SEQ ID NO:Y which havean N-terminus beginning within 5 residues (i.e., + or −5 residues) ofthe predicted cleavage point. Similarly, it is also recognized that insome cases, cleavage of the signal sequence from a secreted protein isnot entirely uniform, resulting in more than one secreted species. Thesepolypeptides, and the polynucleotides encoding such polypeptides, arecontemplated by the present invention.

[0233] Moreover, the signal sequence identified by the above analysismay not necessarily predict the naturally occurring signal sequence. Forexample, the naturally occurring signal sequence may be further upstreamfrom the predicted signal sequence. However, it is likely that thepredicted signal sequence will be capable of directing the secretedprotein to the ER. These polypeptides, and the polynucleotides encodingsuch polypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

[0234] “Variant” refers to a polynucleotide or polypeptide differingfrom the polynucleotide or polypeptide of the present invention, butretaining essential properties thereof. Generally, variants are overallclosely similar, and, in many regions, identical to the polynucleotideor polypeptide of the present invention.

[0235] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence of thepresent invention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding thepolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence shown in Table 1, the ORF (open reading frame), orany fragement specified as described herein.

[0236] As a practical matter, whether any particular nucleic acidmolecule or polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence of the presence invention can bedetermined conventionally using known computer programs. A preferredmethod for determing the best overall match between a query sequence (asequence of the present invention) and a subject sequence, also referredto as a global sequence alignment, can be determined using the FASTDBcomputer program based on the algorithm of Brutlag et al. (Comp. App.Biosci. (1990) 6:237-245). In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byconverting U's to T's. The result of said global sequence alignment isin percent identity. Preferred parameters used in a FASTDB alignment ofDNA sequences to calculate percent identiy are: Matrix=Unitary,k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization GroupLength=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, WindowSize=500 or the lenght of the subject nucleotide sequence, whichever isshorter.

[0237] If the subject sequence is shorter than the query sequencebecause of 5′ or 3′ deletions, not because of internal deletions, amanual correction must be made to the results. This is because theFASTDB program does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.Whether a nucleotide is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score is what is used for the purposes of the presentinvention. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score.

[0238] For example, a 90 base subject sequence is aligned to a 100 basequery sequence to determine percent identity. The deletions occur at the5′ end of the subject sequence and therefore, the FASTDB alignment doesnot show a matched/alignement of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequnce aremanually corrected for. No other manual corrections are to made for thepurposes of the, present invention.

[0239] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a query amino acid sequence of the presentinvention, it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a query amino acid sequence, up to 5% of the amino acidresidues in the subject sequence may be inserted, deleted, (indels) orsubstituted with another amino acid. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0240] As a practical matter, whether any particular polypeptide is atleast 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequences shown in Table 1 or to the amino acid sequenceencoded by deposited DNA clone can be determined conventionally usingknown computer programs. A preferred method for determing the bestoverall match between a query sequence (a sequence of the presentinvention) and a subject sequence, also referred to as a global sequencealignment, can be determined using the FASTDB computer program based onthe algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245).In a sequence alignment the query and subject sequences are either bothnucleotide sequences or both amino acid sequences. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2,Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0,Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

[0241] If the subject sequence is shorter than the query sequence due toN- or C-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is becuase the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

[0242] For example, a 90 amino acid residue subject sequence is alignedwith a 100 residue query sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequnce aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

[0243] The variants may contain alterations in the coding regions,non-coding regions, or both. Especially preferred are polynucleotidevariants containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Nucleotide variants produced by silentsubstitutions due to the degeneracy of the genetic code are preferred.Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.Polynucleotide variants can be produced for a variety of reasons, e.g.,to optimize codon expression for a particular host (change codons in thehuman mRNA to those preferred by a bacterial host such as E. coli).

[0244] Naturally occurring variants are called “allelic variants,” andrefer to one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985).) These allelic variants can vary ateither the polynucleotide and/or polypeptide level. Alternatively,non-naturally occurring variants may be produced by mutagenesistechniques or by direct synthesis.

[0245] Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the secreted protein without substantial loss ofbiological function. The authors of Ron et al., J. Biol. Chem. 268:2984-2988 (1993), reported variant KGF proteins having heparin bindingactivity even after deleting 3, 8, or 27 amino-terminal amino acidresidues. Similarly, Interferon gamma exhibited up to ten times higheractivity after deleting 8-10 amino acid residues from the carboxyterminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216(1988).)

[0246] Moreover, ample evidence demonstrates that variants often retaina biological activity similar to that of the naturally occurringprotein. For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111(1993)) conducted extensive mutational analysis of human cytokine IL-la.They used random mutagenesis to generate over 3,500 individual IL-lamutants that averaged 2.5 amino acid changes per variant over the entirelength of the molecule. Multiple mutations were examined at everypossible amino acid position. The investigators found that “[m]ost ofthe molecule could be altered with little effect on either [binding orbiological activity].” (See, Abstract.) In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

[0247] Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

[0248] Thus, the invention further includes polypeptide variants whichshow substantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., Science247:1306-1310 (1990), wherein the authors indicate that there are twomain strategies for studying the tolerance of an amino acid sequence tochange.

[0249] The first strategy exploits the tolerance of amino acidsubstitutions by natural selection during the process of evolution. Bycomparing amino acid sequences in different species, conserved aminoacids can be identified. These conserved amino acids are likelyimportant for protein function. In contrast, the amino acid positionswhere substitutions have been tolerated by natural selection indicatesthat these positions are not critical for protein function. Thus,positions tolerating amino acid substitution could be modified whilestill maintaining biological activity of the protein.

[0250] The second strategy uses genetic engineering to introduce aminoacid changes at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

[0251] As the authors state, these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,most buried (within the tertiary structure of the protein) amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Moreover, tolerated conservativeamino acid substitutions involve replacement of the aliphatic orhydrophobic amino acids Ala, Val, Leu and Ile; replacement of thehydroxyl residues Ser and Thr; replacement of the acidic residues Aspand Glu; replacement of the amide residues Asn and Gln, replacement ofthe basic residues Lys, Arg, and His; replacement of the aromaticresidues Phe, Tyr, and Trp, and replacement of the small-sized aminoacids Ala, Ser, Thr, Met, and Gly.

[0252] Besides conservative amino acid substitution, variants of thepresent invention include (i) substitutions with one or more of thenon-conserved amino acid residues, where the substituted amino acidresidues may or may not be one encoded by the genetic code, or (ii)substitution with one or more of amino acid residues having asubstituent group, or (iii) fusion of the mature polypeptide withanother compound, such as a compound to increase the stability and/orsolubility of the polypeptide (for example, polyethylene glycol), or(iv) fusion of the polypeptide with additional amino acids, such as anIgG Fc fusion region peptide, or leader or secretory sequence, or asequence facilitating purification. Such variant polypeptides are deemedto be within the scope of those skilled in the art from the teachingsherein.

[0253] For example, polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

[0254] A further embodiment of the invention relates to a polypeptidewhich comprises the amino acid sequence of the present invention havingan amino acid sequence which contains at least one amino acidsubstitution, but not more than 50 amino acid substitutions, even morepreferably, not more than 40 amino acid substitutions, still morepreferably, not more than 30 amino acid substitutions, and still evenmore preferably, not more than 20 amino acid substitutions. Of course,in order of ever-increasing preference, it is highly preferable for apolypeptide to have an amino acid sequence which comprises the aminoacid sequence of the present invention, which contains at least one, butnot more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.In specific embodiments, the number of additions, substitutions, and/ordeletions in the amino acid sequence of the present invention orfragments thereof (e.g., the mature form and/or other fragmentsdescribed herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150,conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

[0255] In the present invention, a “polynucleotide fragment” refers to ashort polynucleotide having a nucleic acid sequence contained in thedeposited clone or shown in SEQ ID NO:X. The short nucleotide fragmentsare preferably at least about 15 nt, and more preferably at least about20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length. A fragment “at least 20 ntin length,” for example, is intended to include 20 or more contiguousbases from the cDNA sequence contained in the deposited clone or thenucleotide sequence shown in SEQ ID NO:X. These nucleotide fragments areuseful as diagnostic probes and primers as discussed herein. Of course,larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) arepreferred.

[0256] Moreover, representative examples of polynucleotide fragments ofthe invention, include, for example, fragments having a sequence fromabout nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250,251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700,701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050,1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350,1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650,1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950,1951-2000, or 2001 to the end of SEQ ID NO:X or the cDNA contained inthe deposited clone. In this context “about” includes the particularlyrecited ranges, larger or smaller by several (5, 4, 3, 2, or 1)nucleotides, at either terminus or at both termini. Preferably, thesefragments encode a polypeptide which has biological activity. Morepreferably, these polynucleotides can be used as probes or primers asdiscussed herein.

[0257] In the present invention, a “polypeptide fragment” refers to ashort amino acid sequence contained in SEQ ID NO:Y or encoded by thecDNA contained in the deposited clone. Protein fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 tothe end of the coding region. Moreover, polypeptide fragments can beabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150amino acids in length. In this context “about” includes the particularlyrecited ranges, larger or smaller by several (5, 4, 3, 2, or 1) aminoacids, at either extreme or at both extremes.

[0258] Preferred polypeptide fragments include the secreted protein aswell as the mature form. Further preferred polypeptide fragments includethe secreted protein or the mature form having a continuous series ofdeleted residues from the amino or the carboxy terminus, or both. Forexample, any number of amino acids, ranging from 1-60, can be deletedfrom the amino terminus of either the secreted polypeptide or the matureform. Similarly, any number of amino acids, ranging from 1-30, can bedeleted from the carboxy terminus of the secreted protein or matureform. Furthermore, any combination of the above amino and carboxyterminus deletions are preferred. Similarly, polynucleotide fragmentsencoding these polypeptide fragments are also preferred.

[0259] Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:Y falling withinconserved domains are specifically contemplated by the presentinvention. Moreover, polynucleotide fragments encoding these domains arealso contemplated.

[0260] Other preferred fragments are biologically active fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the polypeptide of thepresent invention. The biological activity of the fragments may includean improved desired activity, or a decreased undesirable activity.

Epitopes & Antibodies

[0261] In the present invention, “epitopes” refer to polypeptidefragments having antigenic or immunogenic activity in an animal,especially in a human. A preferred embodiment of the present inventionrelates to a polypeptide fragment comprising an epitope, as well as thepolynucleotide encoding this fragment. A region of a protein molecule towhich an antibody can bind is defined as an “antigenic epitope.” Incontrast, an “immunogenic epitope” is defined as a part of a proteinthat elicits an antibody response. (See, for instance, Geysen et al.,Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).)

[0262] Fragments which function as epitopes may be produced by anyconventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci.USA 82:5131-5135 (1985) further described in U.S. Pat. No. 4,631,211.)

[0263] In the present invention, antigenic epitopes preferably contain asequence of at least seven, more preferably at least nine, and mostpreferably between about 15 to about 30 amino acids. Antigenic epitopesare useful to raise antibodies, including monoclonal antibodies, thatspecifically bind the epitope. (See, for instance, Wilson et al., Cell37:767-778 (1984); Sutcliffe, J. G. et al., Science 219:660-666 (1983).)

[0264] Similarly, immunogenic epitopes can be used to induce antibodiesaccording to methods well known in the art. (See, for instance,Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al., Proc.Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J. Gen.Virol. 66:2347-2354 (1985).) A preferred immunogenic epitope includesthe secreted protein. The immunogenic epitopes may be presented togetherwith a carrier protein, such as an albumin, to an animal system (such asrabbit or mouse) or, if it is long enough (at least about 25 aminoacids), without a carrier. However, immunogenic epitopes comprising asfew as 8 to 10 amino acids have been shown to be sufficient to raiseantibodies capable of binding to, at the very least, linear epitopes ina denatured polypeptide (e.g., in Western blotting.)

[0265] As used herein, the term “antibody” (Ab) or “monoclonal antibody”(Mab) is meant to include intact molecules as well as antibody fragments(such as, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to protein. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody.(Wahl et al., J. Nucl. Med. 24:316-325 (1983).) Thus, these fragmentsare preferred, as well as the products of a FAB or other immunoglobulinexpression library. Moreover, antibodies of the present inventioninclude chimeric, single chain, and humanized antibodies.

Fusion Proteins

[0266] Any polypeptide of the present invention can be used to generatefusion proteins. For example, the polypeptide of the present invention,when fused to a second protein, can be used as an antigenic tag.Antibodies raised against the polypeptide of the present invention canbe used to indirectly detect the second protein by binding to thepolypeptide. Moreover, because secreted proteins target cellularlocations based on trafficking signals, the polypeptides of the presentinvention can be used as targeting molecules once fused to otherproteins.

[0267] Examples of domains that can be fused to polypeptides of thepresent invention include not only heterologous signal sequences, butalso other heterologous functional regions. The fusion does notnecessarily need to be direct, but may occur through linker sequences.

[0268] Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Also, peptide moieties may be added tothe polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art.

[0269] Moreover, polypeptides of the present invention, includingfragments, and specifically epitopes, can be combined with parts of theconstant domain of immunoglobulins (IgG), resulting in chimericpolypeptides. These fusion proteins facilitate purification and show anincreased half-life in vivo. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP A 394,827; Trauneckeret al., Nature 331:84-86 (1988).) Fusion proteins havingdisulfide-linked dimeric structures (due to the IgG) can also be moreefficient in binding and neutralizing other molecules, than themonomeric secreted protein or protein fragment alone. (Fountoulakis etal., J. Biochem. 270:3958-3964 (1995).)

[0270] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immuno globulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

[0271] Moreover, the polypeptides of the present invention can be fusedto marker sequences, such as a peptide which facilitates purification ofthe fused polypeptide. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Another peptide tag useful for purification, the “HA”tag, corresponds to an epitope derived from the influenza hemagglutininprotein. (Wilson et al., Cell 37:767 (1984).)

[0272] Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

[0273] The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

[0274] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0275] The polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination, and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the transcripts expressed by the constructs willpreferably include a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

[0276] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

[0277] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Other suitable vectors will be readily apparent to the skilled artisan.

[0278] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

[0279] A polypeptide of this invention can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

[0280] Polypeptides of the present invention, and preferably thesecreted form, can also be recovered from: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect, and mammalian cells. Depending upon thehost employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated or may be non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

[0281] In addition to encompassing host cells containing the vectorconstructs discussed herein, the invention also encompasses primary,secondary, and immortalized host cells of vertebrate origin,particularly mammalian origin, that have been engineered to delete orreplace endogenous genetic material (e.g., coding sequence), and/or toinclude genetic material (e.g., heterologous polynucleotide sequences)that is operably associated with the polynucleotides of the invention,and which activates, alters, and/or amplifies endogenouspolynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous polynucleotide sequences via homologousrecombination (see, e.g., U.S. Pat. No. 5.641,670, issued Jun. 24, 1997;International Publication No. WO 96/29411, published Sep. 26, 1996;International Publication No. WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989), the disclosures of each ofwhich are incorporated by reference in their entireties).

Uses of the Polynucleotides

[0282] Each of the polynucleotides identified herein can be used innumerous ways as reagents. The following description should beconsidered exemplary and utilizes known techniques.

[0283] The polynucleotides of the present invention are useful forchromosome identification. There exists an ongoing need to identify newchromosome markers, since few chromosome marking reagents, based onactual sequence data (repeat polymorphisms), are presently available.Each polynucleotide of the present invention can be used as a chromosomemarker.

[0284] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:X.Primers can be selected using computer analysis so that primers do notspan more than one predicted exon in the genomic DNA. These primers arethen used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the SEQ ID NO:X will yield an amplified fragment.

[0285] Similarly, somatic hybrids provide a rapid method of PCR mappingthe polynucleotides to particular chromosomes. Three or more clones canbe assigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, and preselection by hybridization to constructchromosome specific-cDNA libraries.

[0286] Precise chromosomal location of the polynucleotides can also beachieved using fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

[0287] For chromosome mapping, the polynucleotides can be usedindividually (to mark a single chromosome or a single site on thatchromosome) or in panels (for marking multiple sites and/or multiplechromosomes). Preferred polynucleotides correspond to the noncodingregions of the cDNAs because the coding sequences are more likelyconserved within gene families, thus increasing the chance of crosshybridization during chromosomal mapping.

[0288] Once a polynucleotide has been mapped to a precise chromosomallocation, the physical position of the polynucleotide can be used inlinkage analysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library).) Assuming 1 megabase mapping resolution and onegene per 20 kb, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50-500 potential causativegenes.

[0289] Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected individuals can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected individuals, but not in normalindividuals, indicates that the mutation may cause the disease. However,complete sequencing of the polypeptide and the corresponding gene fromseveral normal individuals is required to distinguish the mutation froma polymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

[0290] Furthermore, increased or decreased expression of the gene inaffected individuals as compared to unaffected individuals can beassessed using polynucleotides of the present invention. Any of thesealterations (altered expression, chromosomal rearrangement, or mutation)can be used as a diagnostic or prognostic marker.

[0291] In addition to the foregoing, a polynucleotide can be used tocontrol gene expression through triple helix formation or antisense DNAor RNA. Both methods rely on binding of the polynucleotide to DNA orRNA. For these techniques, preferred polynucleotides are usually 20 to40 bases in length and complementary to either the region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat disease.

[0292] Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell.

[0293] The polynucleotides are also useful for identifying individualsfrom minute biological samples. The United States military, for example,is considering the use of restriction fragment length polymorphism(RFLP) for identification of its personnel. In this technique, anindividual's genormic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentifying personnel. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The polynucleotides of the presentinvention can be used as additional DNA markers for RFLP.

[0294] The polynucleotides of the present invention can also be used asan alternative to RFLP, by determining the actual base-by-base DNAsequence of selected portions of an individual's genome. These sequencescan be used to prepare PCR primers for amplifying and isolating suchselected DNA, which can then be sequenced. Using this technique,individuals can be identified because each individual will have a uniqueset of DNA sequences. Once an unique ID database is established for anindividual, positive identification of that individual, living or dead,can be made from extremely small tissue samples.

[0295] Forensic biology also benefits from using DNA-basedidentification techniques as disclosed herein. DNA sequences taken fromvery small biological samples such as tissues, e.g., hair or skin, orbody fluids, e.g., blood, saliva, semen, etc., can be amplified usingPCR. In one prior art technique, gene sequences amplified frompolymorphic loci, such as DQa class II HLA gene, are used in forensicbiology to identify individuals. (Erlich, H., PCR Technology, Freemanand Co. (1992).) Once these specific polymorphic loci are amplified,they are digested with one or more restriction enzymes, yielding anidentifying set of bands on a Southern blot probed with DNAcorresponding to the DQa class II HLA gene. Similarly, polynucleotidesof the present invention can be used as polymorphic markers for forensicpurposes.

[0296] There is also a need for reagents capable of identifying thesource of a particular tissue. Such need arises, for example, inforensics when presented with tissue of unknown origin. Appropriatereagents can comprise, for example, DNA probes or primers specific toparticular tissue prepared from the sequences of the present invention.Panels of such reagents can identify tissue by species and/or by organtype. In a similar fashion, these reagents can be used to screen tissuecultures for contamination.

[0297] In the very least, the polynucleotides of the present inventioncan be used as molecular weight markers on Southern gels, as diagnosticprobes for the presence of a specific mRNA in a particular cell type, asa probe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

Uses of the Polypeptides

[0298] Each of the polypeptides identified herein can be used innumerous ways. The following description should be considered exemplaryand utilizes known techniques.

[0299] A polypeptide of the present invention can be used to assayprotein levels in a biological sample using antibody-based techniques.For example, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125 I, 121 I), carbon (14C), sulfur (35 S), tritium (3 H), indium (112 In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, andbiotin.

[0300] In addition to assaying secreted protein levels in a biologicalsample, proteins can also be detected in vivo by imaging. Antibodylabels or markers for in vivo imaging of protein include thosedetectable by X-radiography, NMR or ESR. For X-radiography, suitablelabels include radioisotopes such as barium or cesium, which emitdetectable radiation but are not overtly harmful to the subject.Suitable markers for NMR and ESR include those with a detectablecharacteristic spin, such as deuterium, which may be incorporated intothe antibody by labeling of nutrients for the relevant hybridoma.

[0301] A protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, 131 I, 112 In, 99 mTc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously, orintraperitoneally) into the mammal. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of 99 mTc. The labeled antibody or antibody fragment willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).)

[0302] Thus, the invention provides a diagnostic method of a disorder,which involves (a) assaying the expression of a polypeptide of thepresent invention in cells or body fluid of an individual; (b) comparingthe level of gene expression with a standard gene expression level,whereby an increase or decrease in the assayed polypeptide geneexpression level compared to the standard expression level is indicativeof a disorder.

[0303] Moreover, polypeptides of the present invention can be used totreat disease. For example, patients can be administered a polypeptideof the present invention in an effort to replace absent or decreasedlevels of the polypeptide (e.g., insulin), to supplement absent ordecreased levels of a different polypeptide (e.g., hemoglobin S forhemoglobin B), to inhibit the activity of a polypeptide (e.g., anoncogene), to activate the activity of a polypeptide (e.g., by bindingto a receptor), to reduce the activity of a membrane bound receptor bycompeting with it for free ligand (e.g., soluble TNF receptors used inreducing inflammation), or to bring about a desired response (e.g.,blood vessel growth).

[0304] Similarly, antibodies directed to a polypeptide of the presentinvention can also be used to treat disease. For example, administrationof an antibody directed to a polypeptide of the present invention canbind and reduce overproduction of the polypeptide. Similarly,administration of an antibody can activate the polypeptide, such as bybinding to a polypeptide bound to a membrane (receptor).

[0305] At the very least, the polypeptides of the present invention canbe used as molecular weight markers on SDS-PAGE gels or on molecularsieve gel filtration columns using methods well known to those of skillin the art. Polypeptides can also be used to raise antibodies, which inturn are used to measure protein expression from a recombinant cell, asa way of assessing transformation of the host cell. Moreover, thepolypeptides of the present invention can be used to test the followingbiological activities.

Biological Activities

[0306] The polynucleotides and polypeptides of the present invention canbe used in assays to test for one or more biological activities. Ifthese polynucleotides and polypeptides do exhibit activity in aparticular assay, it is likely that these molecules may be involved inthe diseases associated with the biological activity. Thus, thepolynucleotides and polypeptides could be used to treat the associateddisease.

Immune Activity

[0307] A polypeptide or polynucleotide of the present invention may beuseful in treating deficiencies or disorders of the immune system, byactivating or inhibiting the proliferation, differentiation, ormobilization (chemotaxis) of immune cells. Immune cells develop througha process called hematopoiesis, producing myeloid (platelets, red bloodcells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)cells from pluripotent stem cells. The etiology of these immunedeficiencies or disorders may be genetic, somatic, such as cancer orsome autoimmune disorders, acquired (e.g., by chemotherapy or toxins),or infectious. Moreover, a polynucleotide or polypeptide of the presentinvention can be used as a marker or detector of a particular immunesystem disease or disorder.

[0308] A polynucleotide or polypeptide of the present invention may beuseful in treating or detecting deficiencies or disorders ofhematopoietic cells. A polypeptide or polynucleotide of the presentinvention could be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effortto treat those disorders associated with a decrease in certain (or many)types hematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein disorders (e.g.agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, commonvariable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLVinfection, leukocyte adhesion deficiency syndrome, lymphopenia,phagocyte bactericidal dysfunction, severe combined immunodeficiency(SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, orhemoglobinuria.

[0309] Moreover, a polypeptide or polynucleotide of the presentinvention could also be used to modulate hemostatic (the stopping ofbleeding) or thrombolytic activity (clot formation). For example, byincreasing hemostatic or thrombolytic activity, a polynucleotide orpolypeptide of the present invention could be used to treat bloodcoagulation disorders (e.g., afibrinogenemia, factor deficiencies),blood platelet disorders (e.g. thrombocytopenia), or wounds resultingfrom trauma, surgery, or other causes. Alternatively, a polynucleotideor polypeptide of the present invention that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting.These molecules could be important in the treatment of heart attacks(infarction), strokes, or scarring.

[0310] A polynucleotide or polypeptide of the present invention may alsobe useful in treating or detecting autoimmune disorders. Many autoimmunedisorders result from inappropriate recognition of self as foreignmaterial by immune cells. This inappropriate recognition results in animmune response leading to the destruction of the host tissue.Therefore, the administration of a polypeptide or polynucleotide of thepresent invention that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune disorders.

[0311] Examples of autoimmune disorders that can be treated or detectedby the present invention include, but are not limited to: Addison'sDisease, hemolytic anemia, antiphospholipid syndrome, rheumatoidarthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

[0312] Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by a polypeptide or polynucleotide of the present invention.Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

[0313] A polynucleotide or polypeptide of the present invention may alsobe used to treat and/or prevent organ rejection or graft-versus-hostdisease (GVHD). Organ rejection occurs by host immune cell destructionof the transplanted tissue through an immune response. Similarly, animmune response is also involved in GVHD, but, in this case, the foreigntransplanted immune cells destroy the host tissues. The administrationof a polypeptide or polynucleotide of the present invention thatinhibits an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing organ rejection or GVHD.

[0314] Similarly, a polypeptide or polynucleotide of the presentinvention may also be used to modulate inflammation. For example, thepolypeptide or polynucleotide may inhibit the proliferation anddifferentiation of cells involved in an inflammatory response. Thesemolecules can be used to treat inflammatory conditions, both chronic andacute conditions, including inflammation associated with infection(e.g., septic shock, sepsis, or systemic inflammatory response syndrome(SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

[0315] A polypeptide or polynucleotide can be used to treat or detecthyperproliferative disorders, including neoplasms. A polypeptide orpolynucleotide of the present invention may inhibit the proliferation ofthe disorder through direct or indirect interactions. Alternatively, apolypeptide or polynucleotide of the present invention may proliferateother cells which can inhibit the hyperproliferative disorder.

[0316] For example, by increasing an immune response, particularlyincreasing antigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

[0317] Examples of hyperproliferative disorders that can be treated ordetected by a polynucleotide or polypeptide of the present inventioninclude, but are not limited to neoplasms located in the: abdomen, bone,breast, digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous (central and peripheral), lymphatic system,pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

[0318] Similarly, other hyperproliferative disorders can also be treatedor detected by a polynucleotide or polypeptide of the present invention.Examples of such hyperproliferative disorders include, but are notlimited to: hypergammaglobulinemia, lymphoproliferative disorders,paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron'sMacroglobulinemia, Gaucher's Disease, histiocytosis, and any otherhyperproliferative disease, besides neoplasia, located in an organsystem listed above.

Infectious Disease

[0319] A polypeptide or polynucleotide of the present invention can beused to treat or detect infectious agents. For example, by increasingthe immune response, particularly increasing the proliferation anddifferentiation of B and/or T cells, infectious diseases may be treated.The immune response may be increased by either enhancing an existingimmune response, or by initiating a new immune response. Alternatively,the polypeptide or polynucleotide of the present invention may alsodirectly inhibit the infectious agent, without necessarily eliciting animmune response.

[0320] Viruses are one example of an infectious agent that can causedisease or symptoms that can be treated or detected by a polynucleotideor polypeptide of the present invention. Examples of viruses, include,but are not limited to the following DNA and RNA viral families:Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae,Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza),Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such asSmallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae(HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus).Viruses falling within these families can cause a variety of diseases orsymptoms, including, but not limited to: arthritis, bronchiollitis,encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronicfatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt'sLymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza,Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitteddiseases, skin diseases (e.g., Kaposi's, warts), and viremia. Apolypeptide or polynucleotide of the present invention can be used totreat or detect any of these symptoms or diseases.

[0321] Similarly, bacterial or fungal agents that can cause disease orsymptoms and that can be treated or detected by a polynucleotide orpolypeptide of the present invention include, but not limited to, thefollowing Gram-Negative and Gram-positive bacterial families and fungi:Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia),Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae,Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses,Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia),Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria,Mycoplasmatales, Neisseriaceae (e.g., Acinetobacter, Gonorrhea,Menigococcal), Pasteurellacea Infections (e.g., Actinobacillus,Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae,Syphilis, and Staphylococcal. These bacterial or fungal families cancause the following diseases or symptoms, including, but not limited to:bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis,uveitis), gingivitis, opportunistic infections (e.g., AIDS relatedinfections), paronychia, prosthesis-related infections, Reiter'sDisease, respiratory tract infections, such as Whooping Cough orEmpyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery,Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea,meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. A polypeptide or polynucleotide of the presentinvention can be used to treat or detect any of these symptoms ordiseases.

[0322] Moreover, parasitic agents causing disease or symptoms that canbe treated or detected by a polynucleotide or polypeptide of the presentinvention include, but not limited to, the following families:Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis,Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis,Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas. Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), Malaria, pregnancycomplications, and toxoplasmosis. A polypeptide or polynucleotide of thepresent invention can be used to treat or detect any of these symptomsor diseases.

[0323] Preferably, treatment using a polypeptide or polynucleotide ofthe present invention could either be by administering an effectiveamount of a polypeptide to the patient, or by removing cells from thepatient, supplying the cells with a polynucleotide of the presentinvention, and returning the engineered cells to the patient (ex vivotherapy). Moreover, the polypeptide or polynucleotide of the presentinvention can be used as an antigen in a vaccine to raise an immuneresponse against infectious disease.

Regeneration

[0324] A polynucleotide or polypeptide of the present invention can beused to differentiate, proliferate, and attract cells, leading to theregeneration of tissues. (See, Science 276:59-87 (1997).) Theregeneration of tissues could be used to repair, replace, or protecttissue damaged by congenital defects, trauma (wounds, burns, incisions,or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

[0325] Tissues that could be regenerated using the present inventioninclude organs (e.g., pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac), vasculature(including vascular and lymphatics), nervous, hematopoietic, andskeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,regeneration occurs without or decreased scarring. Regeneration also mayinclude angiogenesis.

[0326] Moreover, a polynucleotide or polypeptide of the presentinvention may increase regeneration of tissues difficult to heal. Forexample, increased tendon/ligament regeneration would quicken recoverytime after damage. A polynucleotide or polypeptide of the presentinvention could also be used prophylactically in an effort to avoiddamage. Specific diseases that could be treated include of tendinitis,carpal tunnel syndrome, and other tendon or ligament defects. A furtherexample of tissue regeneration of non-healing wounds includes pressureulcers, ulcers associated with vascular insufficiency, surgical, andtraumatic wounds.

[0327] Similarly, nerve and brain tissue could also be regenerated byusing a polynucleotide or polypeptide of the present invention toproliferate and differentiate nerve cells. Diseases that could betreated using this method include central and peripheral nervous systemdiseases, neuropathies, or mechanical and traumatic disorders (e.g.,spinal cord disorders, head trauma, cerebrovascular disease, and stoke).Specifically, diseases associated with peripheral nerve injuries,peripheral neuropathy (e.g., resulting from chemotherapy or othermedical therapies), localized neuropathies, and central nervous systemdiseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), couldall be treated using the polynucleotide or polypeptide of the presentinvention.

Chemotaxis

[0328] A polynucleotide or polypeptide of the present invention may havechemotaxis activity. A chemotaxic molecule attracts or mobilizes cells(e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells,eosinophils, epithelial and/or endothelial cells) to a particular sitein the body, such as inflammation, infection, or site ofhyperproliferation. The mobilized cells can then fight off and/or healthe particular trauma or abnormality.

[0329] A polynucleotide or polypeptide of the present invention mayincrease chemotaxic activity of particular cells. These chemotacticmolecules can then be used to treat inflammation, infection,hyperproliferative disorders, or any immune system disorder byincreasing the number of cells targeted to a particular location in thebody. For example, chemotaxic molecules can be used to treat wounds andother trauma to tissues by attracting immune cells to the injuredlocation. Chemotactic molecules of the present invention can alsoattract fibroblasts, which can be used to treat wounds.

[0330] It is also contemplated that a polynucleotide or polypeptide ofthe present invention may inhibit chemotactic activity. These moleculescould also be used to treat disorders. Thus, a polynucleotide orpolypeptide of the present invention could be used as an inhibitor ofchemotaxis.

Binding Activity

[0331] A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors), orsmall molecules.

[0332] Preferably, the molecule is closely related to the natural ligandof the polypeptide, e.g., a fragment of the ligand, or a naturalsubstrate, a ligand, a structural or functional mimetic. (See, Coliganet al., Current Protocols in Immunology 1(2):Chapter 5 (1991).)Similarly, the molecule can be closely related to the natural receptorto which the polypeptide binds, or at least, a fragment of the receptorcapable of being bound by the polypeptide (e.g., active site). In eithercase, the molecule can be rationally designed using known techniques.

[0333] Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

[0334] The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

[0335] Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

[0336] Preferably, an ELISA assay can measure polypeptide level oractivity in a sample (e.g., biological sample) using a monoclonal orpolyclonal antibody. The antibody can measure polypeptide leyel oractivity by either binding, directly or indirectly, to the polypeptideor by competing with the polypeptide for a substrate.

[0337] All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting thepolypeptide/molecule. Moreover, the assays can discover agents which mayinhibit or enhance the production of the polypeptide from suitablymanipulated cells or tissues.

[0338] Therefore, the invention includes a method of identifyingcompounds which bind to a polypeptide of the invention comprising thesteps of: (a) incubating a candidate binding compound with a polypeptideof the invention; and (b) determining if binding has occurred. Moreover,the invention includes a method of identifying agonists/antagonistscomprising the steps of: (a) incubating a candidate compound with apolypeptide of the invention, (b) assaying a biological activity, and(b) determining if a biological activity of the polypeptide has beenaltered.

Other Activities

[0339] A polypeptide or polynucleotide of the present invention may alsoincrease or decrease the differentiation or proliferation of embryonicstem cells, besides, as discussed above, hematopoietic lineage.

[0340] A polypeptide or polynucleotide of the present invention may alsobe used to modulate mammalian characteristics, such as body height,weight, hair color, eye color, skin, percentage of adipose tissue,pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, apolypeptide or polynucleotide of the present invention may be used tomodulate mammalian metabolism affecting catabolism, anabolism,processing, utilization, and storage of energy.

[0341] A polypeptide or polynucleotide of the present invention may beused to change a mammal's mental state or physical state by influencingbiorhythms, caricadic rhythms, depression (including depressivedisorders), tendency for violence, tolerance for pain, reproductivecapabilities (preferably by Activin or Innibin-like activity), hormonalor endocrine levels, appetite, libido, memory, stress, or othercognitive qualities.

[0342] A polypeptide or polynucleotide of the present invention may alsobe used as a food additive or preservative, such as to increase ordecrease storage capabilities, fat content, lipid, protein,carbohydrate, vitamins, minerals, cofactors or other nutritionalcomponents.

Other Preferred Embodiments

[0343] Other preferred embodiments of the claimed invention include anisolated nucleic acid molecule comprising a nucleotide sequence which isat least 95% identical to a sequence of at least about 50 contiguousnucleotides in the nucleotide sequence of SEQ ID NO:X wherein X is anyinteger as defined in Table 1.

[0344] Also preferred is a nucleic acid molecule wherein said sequenceof contiguous nucleotides is included in the nucleotide sequence of SEQID NO:X in the range of positions beginning with the nucleotide at aboutthe position of the 5′ Nucleotide of the Clone Sequence and ending withthe nucleotide at about the position of the 3′ Nucleotide of the CloneSequence as defined for SEQ ID NO:X in Table 1.

[0345] Also preferred is a nucleic acid molecule wherein said sequenceof contiguous nucleotides is included in the nucleotide sequence of SEQID NO:X in the range of positions beginning with the nucleotide at aboutthe position of the 5′ Nucleotide of the Start Codon and ending with thenucleotide at about the position of the 3′ Nucleotide of the CloneSequence as defined for SEQ ID NO:X in Table 1.

[0346] Similarly preferred is a nucleic acid molecule wherein saidsequence of contiguous nucleotides is included in the nucleotidesequence of SEQ ID NO:X in the range of positions beginning with thenucleotide at about the position of the 5′ Nucleotide of the First AminoAcid of the Signal Peptide and ending with the nucleotide at about theposition of the 3′ Nucleotide of the Clone Sequence as defined for SEQID NO:X in Table 1.

[0347] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 150 contiguous nucleotides in the nucleotide sequence of SEQID NO:X.

[0348] Further preferred is an isolated nucleic acid molecule comprisinga nucleotide sequence which is at least 95% identical to a sequence ofat least about 500 contiguous nucleotides in the nucleotide sequence ofSEQ ID NO:X.

[0349] A further preferred embodiment is a nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thenucleotide sequence of SEQ ID NO:X beginning with the nucleotide atabout the position of the 5′ Nucleotide of the First Amino Acid of theSignal Peptide and ending with the nucleotide at about the position ofthe 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X inTable 1.

[0350] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to the complete nucleotide sequence of SEQ ID NO:X.

[0351] Also preferred is an isolated nucleic acid molecule whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule, wherein said nucleic acid molecule which hybridizes does nothybridize under stringent hybridization conditions to a nucleic acidmolecule having a nucleotide sequence consisting of only A residues orof only T residues.

[0352] Also preferred is a composition of matter comprising a DNAmolecule which comprises a human cDNA clone identified by a cDNA CloneIdentifier in Table 1, which DNA molecule is contained in the materialdeposited with the American Type Culture Collection and given the ATCCDeposit Number shown in Table 1 for said cDNA Clone Identifier.

[0353] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in the nucleotide sequence of a humancDNA clone identified by a cDNA Clone Identifier in Table 1, which DNAmolecule is contained in the deposit given the ATCC Deposit Number shownin Table 1.

[0354] Also preferred is an isolated nucleic acid molecule, wherein saidsequence of at least 50 contiguous nucleotides is included in thenucleotide sequence of the complete open reading frame sequence encodedby said human cDNA clone.

[0355] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to sequence of atleast 150 contiguous nucleotides in the nucleotide sequence encoded bysaid human cDNA clone.

[0356] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to sequence of at least 500 contiguous nucleotides in thenucleotide sequence encoded by said human cDNA clone.

[0357] A further preferred embodiment is an isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to the complete nucleotide sequence encoded by said human cDNAclone.

[0358] A further preferred embodiment is a method for detecting in abiological sample a nucleic acid molecule comprising a nucleotidesequence which is at least 95% identical to a sequence of at least 50contiguous nucleotides in a sequence selected from the group consistingof: a nucleotide sequence of SEQ ID NO:X wherein X is any integer asdefined in Table 1; and a nucleotide sequence encoded by a human cDNAclone identified by a cDNA Clone Identifier in Table 1 and contained inthe deposit with the ATCC Deposit Number shown for said cDNA clone inTable 1; which method comprises a step of comparing a nucleotidesequence of at least one nucleic acid molecule in said sample with asequence selected from said group and determining whether the sequenceof said nucleic acid molecule in said sample is at least 95% identicalto said selected sequence.

[0359] Also preferred is the above method wherein said step of comparingsequences comprises determining the extent of nucleic acid hybridizationbetween nucleic acid molecules in said sample and a nucleic acidmolecule comprising said sequence selected from said group. Similarly,also preferred is the above method wherein said step of comparingsequences is performed by comparing the nucleotide sequence determinedfrom a nucleic acid molecule in said sample with said sequence selectedfrom said group. The nucleic acid molecules can comprise DNA moleculesor RNA molecules.

[0360] A further preferred embodiment is a method for identifying thespecies, tissue or cell type of a biological sample which methodcomprises a step of detecting nucleic acid molecules in said sample, ifany, comprising a nucleotide sequence that is at least 95% identical toa sequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:Xwherein X is any integer as defined in Table 1; and a nucleotidesequence encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1.

[0361] The method for identifying the species, tissue or cell type of abiological sample can comprise a step of detecting nucleic acidmolecules comprising a nucleotide sequence in a panel of at least twonucleotide sequences, wherein at least one sequence in said panel is atleast 95% identical to a sequence of at least 50 contiguous nucleotidesin a sequence selected from said group.

[0362] Also preferred is a method for diagnosing in a subject apathological condition associated with abnormal structure or expressionof a gene encoding a secreted protein identified in Table 1, whichmethod comprises a step of detecting in a biological sample obtainedfrom said subject nucleic acid molecules, if any, comprising anucleotide sequence that is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from the groupconsisting of: a nucleotide sequence of SEQ ID NO:X wherein X is anyinteger as defined in Table 1; and a nucleotide sequence encoded by ahuman cDNA clone identified by a cDNA Clone Identifier in Table 1 andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table 1.

[0363] The method for diagnosing a pathological condition can comprise astep of detecting nucleic acid molecules comprising a nucleotidesequence in a panel of at least two nucleotide sequences, wherein atleast one sequence in said panel is at least 95% identical to a sequenceof at least 50 contiguous nucleotides in a sequence selected from saidgroup.

[0364] Also preferred is a composition of matter comprising isolatednucleic acid molecules wherein the nucleotide sequences of said nucleicacid molecules comprise a panel of at least two nucleotide sequences,wherein at least one sequence in said panel is at least 95% identical toa sequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:Xwherein X is any integer as defined in Table 1; and a nucleotidesequence encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1. The nucleic acid moleculescan comprise DNA molecules or RNA molecules.

[0365] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the amino acid sequence of SEQ ID NO:Y whereinY is any integer as defined in Table 1.

[0366] Also preferred is a polypeptide, wherein said sequence ofcontiguous amino acids is included in the amino acid sequence of SEQ IDNO:Y in the range of positions beginning with the residue at about theposition of the First Amino Acid of the Secreted Portion and ending withthe residue at about the Last Amino Acid of the Open Reading Frame asset forth for SEQ ID NO:Y in Table 1.

[0367] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

[0368] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

[0369] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to the complete amino acid sequenceof SEQ ID NO:Y.

[0370] Further preferred is an isolated polypeptide comprising an aminoacid sequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the complete amino acid sequence of a secretedprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1.

[0371] Also preferred is a polypeptide wherein said sequence ofcontiguous amino acids is included in the amino acid sequence of asecreted portion of the secreted protein encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1 and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1.

[0372] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of the secretedportion of the protein encoded by a human cDNA clone identified by acDNA Clone Identifier in Table 1 and contained in the deposit with theATCC Deposit Number shown for said cDNA clone in Table 1.

[0373] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of the secretedportion of the protein encoded by a human cDNA clone identified by acDNA Clone Identifier in Table 1 and contained in the deposit with theATCC Deposit Number shown for said cDNA clone in Table 1.

[0374] Also preferred is an isolated polypeptide comprising an aminoacid sequence at least 95% identical to the amino acid sequence of thesecreted portion of the protein encoded by a human cDNA clone identifiedby a cDNA Clone Identifier in Table 1 and contained in the deposit withthe ATCC Deposit Number shown for said cDNA clone in Table 1.

[0375] Further preferred is an isolated antibody which bindsspecifically to a polypeptide comprising an amino acid sequence that isat least 90% identical to a sequence of at least 10 contiguous aminoacids in a sequence selected from the group consisting of: an amino acidsequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1;and a complete amino acid sequence of a protein encoded by a human cDNAclone identified by a cDNA Clone Identifier in Table 1 and contained inthe deposit with the ATCC Deposit Number shown for said cDNA clone inTable 1.

[0376] Further preferred is a method for detecting in a biologicalsample a polypeptide comprising an amino acid sequence which is at least90% identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and acomplete amino acid sequence of a protein encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1 and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1; which method comprises a step of comparing an amino acid sequence ofat least one polypeptide molecule in said sample with a sequenceselected from said group and determining whether the sequence of saidpolypeptide molecule in said sample is at least 90% identical to saidsequence of at least 10 contiguous amino acids.

[0377] Also preferred is the above method wherein said step of comparingan amino acid sequence of at least one polypeptide molecule in saidsample with a sequence selected from said group comprises determiningthe extent of specific binding of polypeptides in said sample to anantibody which binds specifically to a polypeptide comprising an aminoacid sequence that is at least 90% identical to a sequence of at least10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1; and a complete amino acid sequence of aprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1 and contained in the deposit with the ATCC DepositNumber shown for said cDNA clone in Table 1.

[0378] Also preferred is the above method wherein said step of comparingsequences is performed by comparing the amino acid sequence determinedfrom a polypeptide molecule in said sample with said sequence selectedfrom said group.

[0379] Also preferred is a method for identifying the species, tissue orcell type of a biological sample which method comprises a step ofdetecting polypeptide molecules in said sample, if any, comprising anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1; and a complete amino acid sequence of asecreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table 1 and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1.

[0380] Also preferred is the above method for identifying the species,tissue or cell type of a biological sample, which method comprises astep of detecting polypeptide molecules comprising an amino acidsequence in a panel of at least two amino acid sequences, wherein atleast one sequence in said panel is at least 90% identical to a sequenceof at least 10 contiguous amino acids in a sequence selected from theabove group.

[0381] Also preferred is a method for diagnosing in a subject apathological condition associated with abnormal structure or expressionof a gene encoding a secreted protein identified in Table 1, whichmethod comprises a step of detecting in a biological sample obtainedfrom said subject polypeptide molecules comprising an amino acidsequence in a panel of at least two amino acid sequences, wherein atleast one sequence in said panel is at least 90% identical to a sequenceof at least 10 contiguous amino acids in a sequence selected from thegroup consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y isany integer as defined in Table 1; and a complete amino acid sequence ofa secreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table 1 and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1.

[0382] In any of these methods, the step of detecting said polypeptidemolecules includes using an antibody.

[0383] Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a nucleotidesequence encoding a polypeptide wherein said polypeptide comprises anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1; and a complete amino acid sequence of asecreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table 1 and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1.

[0384] Also preferred is an isolated nucleic acid molecule, wherein saidnucleotide sequence encoding a polypeptide has been optimized forexpression of said polypeptide in a prokaryotic host.

[0385] Also preferred is an isolated nucleic acid molecule, wherein saidpolypeptide comprises an amino acid sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1; and a complete amino acid sequence of asecreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table 1 and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1.

[0386] Further preferred is a method of making a recombinant vectorcomprising inserting any of the above isolated nucleic acid moleculeinto a vector. Also preferred is the recombinant vector produced by thismethod. Also preferred is a method of making a recombinant host cellcomprising introducing the vector into a host cell, as well as therecombinant host cell produced by this method.

[0387] Also preferred is a method of making an isolated polypeptidecomprising culturing this recombinant host cell under conditions suchthat said polypeptide is expressed and recovering said polypeptide. Alsopreferred is this method of making an isolated polypeptide, wherein saidrecombinant host cell is a eukaryotic cell and said polypeptide is asecreted portion of a human secreted protein comprising an amino acidsequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y beginning with the residue at the position of the FirstAmino Acid of the Secreted Portion of SEQ ID NO:Y wherein Y is aninteger set forth in Table 1 and said position of the First Amino Acidof the Secreted Portion of SEQ ID NO:Y is defined in Table 1; and anamino acid sequence of a secreted portion of a protein encoded by ahuman cDNA clone identified by a cDNA Clone Identifier in Table 1 andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table 1. The isolated polypeptide produced by this methodis also preferred.

[0388] Also preferred is a method of treatment of an individual in needof an increased level of a secreted protein activity, which methodcomprises administering to such an individual a pharmaceuticalcomposition comprising an amount of an isolated polypeptide,polynucleotide, or antibody of the claimed invention effective toincrease the level of said protein activity in said individual.

[0389] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1 Isolation of a Selected cDNA Clone From the DepositedSample

[0390] Each cDNA clone in a cited ATCC deposit is contained in a plasmidvector. Table 1 identifies the vectors used to construct the cDNAlibrary from which each clone was isolated. In many cases, the vectorused to construct the library is a phage vector from which a plasmid hasbeen excised. The table immediately below correlates the related plasmidfor each phage vector used in constructing the cDNA library. Forexample, where a particular clone is identified in Table 1 as beingisolated in the vector “Lambda Zap,” the corresponding deposited cloneis in “pBluescript.” Vector Used to Construct Library CorrespondingDeposited Plasmid Lambda Zap pBluescript (pBS) Uni-Zap XR pBluescript(pBS) Zap Express pBK lafmid BA plafmid BA pSport1 pSport1 pCMVSport 2.0pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0 pCR ® 2.1 pCR ® 2.1

[0391] Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636),Uni-Zap XR (U.S. Pat. Nos. 5,128, 256 and 5,286,636), Zap Express (U.S.Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. etal., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. andShort, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees,M. A. et al., Strategies 5:58-61 (1992)) are commercially available fromStratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla,Calif., 92037. pBS contains an ampicillin resistance gene and pBKcontains a neomycin resistance gene. Both can be transformed into E.coli strain XL-1 Blue, also available from Stratagene. pBS comes in 4forms SK+, SK−, KS+ and KS. The S and K refers to the orientation of thepolylinker to the T7 and T3 primer sequences which flank the polylinkerregion (“S” is for SacI and “K” is for KpnI which are the first sites oneach respective end of the linker). “+” or “−” refer to the orientationof the f1 origin of replication (“ori”), such that in one orientation,single stranded rescue initiated from the f1 ori generates sense strandDNA and in the other, antisense.

[0392] Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtainedfrom Life Technologies, Inc., P. O. Box 6009, Gaithersburg, Md. 20897.All Sport vectors contain an ampicillin resistance gene and may betransformed into E. coli strain DH10B, also available from LifeTechnologies. (See, for instance, Gruber, C. E., et al., Focus 15:59(1993).) Vector lafmid BA (Bento Soares, Columbia University, NY)contains an ampicillin resistance gene and can be transformed into E.coli strain XL-1 Blue. Vector pCR®2.1, which is available fromInvitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains anampicillin resistance gene and may be transformed into E. coli strainDH10B, available from Life Technologies. (See, for instance, Clark, J.M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al.,Bio/Technology 9: (1991).) Preferably, a polynucleotide of the presentinvention does not comprise the phage vector sequences identified forthe particular clone in Table 1, as well as the corresponding plasmidvector sequences designated above.

[0393] The deposited material in the sample assigned the ATCC DepositNumber cited in Table 1 for any given cDNA clone also may contain one ormore additional plasmids, each comprising a cDNA clone different fromthat given clone. Thus, deposits sharing the same ATCC Deposit Numbercontain at least a plasmid for each cDNA clone identified in Table 1.Typically, each ATCC deposit sample cited in Table 1 comprises a mixtureof approximately equal amounts (by weight) of about 50 plasmid DNAs,each containing a different cDNA clone; but such a deposit sample mayinclude plasmids for more or less than 50 cDNA clones, up to about 500cDNA clones.

[0394] Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNAs cited for that clone in Table 1. First,a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO:X.

[0395] Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with³²P-γ-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, NY (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

[0396] Alternatively, two primers of 17-20 nucleotides derived from bothends of the SEQ ID NO:X (i.e., within the region of SEQ ID NO:X boundedby the 5′ NT and the 3′ NT of the clone defined in Table 1) aresynthesized and used to amplify the desired cDNA using the depositedcDNA plasmid as a template. The polymerase chain reaction is carried outunder routine conditions, for instance, in 25 μl of reaction mixturewith 0.5 ug of the above cDNA template. A convenient reaction mixture is1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP,dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirtyfive cycles of PCR (denaturation at 94° C. for 1 min; annealing at 55° Cfor 1 min; elongation at 72° C. for 1 min) are performed with aPerkin-Elmer Cetus automated thermal cycler. The amplified product isanalyzed by agarose gel electrophoresis and the DNA band with expectedmolecular weight is excised and purified. The PCR product is verified tobe the selected sequence by subcloning and sequencing the DNA product.

[0397] Several methods are available for the identification of the 5′ or3′ non-coding portions of a gene which may not be present in thedeposited clone. These methods include but are not limited to, filterprobing, clone enrichment using specific probes, and protocols similaror identical to 5′ and 3′ “RACE” protocols which are well known in theart. For instance, a method similar to 5′ RACE is available forgenerating the missing 5′ end of a desired full-length transcript.(Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993).)

[0398] Briefly, a specific RNA oligonucleotide is ligated to the 5′ endsof a population of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full length gene.

[0399] This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA which may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

[0400] This modified RNA preparation is used as a template for firststrand cDNA synthesis using a gene specific oligonucleotide. The firststrand synthesis reaction is used as a template for PCR amplification ofthe desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene.

Example 2 Isolation of Genomic Clones Corresponding to a Polynucleotide

[0401] A human genomic P1 library (Genomic Systems, Inc.) is screened byPCR using primers selected for the cDNA sequence corresponding to SEQ IDNO:X., according to the method described in Example 1. (See also,Sambrook.)

Example 3 Tissue Distribution of Polypeptide

[0402] Tissue distribution of mRNA expression of polynucleotides of thepresent invention is determined using protocols for Northern blotanalysis, described by, among others, Sambrook et al. For example, acDNA probe produced by the method described in Example 1 is labeled withP³² using the rediprime™ DNA labeling system (Amersham Life Science),according to manufacturer's instructions. After labeling, the probe ispurified using CHROMA SPIN-100™ column (Clontech Laboratories, Inc.),according to manufacturer's protocol number PT1200-1. The purifiedlabeled probe is then used to examine various human tissues for mRNAexpression.

[0403] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IM) (Clontech) are examinedwith the labeled probe using ExpressHyb™ hybridization solution(Clontech) according to manufacturer's protocol number PT1190-1.Following hybridization and washing, the blots are mounted and exposedto film at −70° C. overnight, and the films developed according tostandard procedures.

Example 4 Chromosomal Mapping of the Polynucleotides

[0404] An oligonucleotide primer set is designed according to thesequence at the 5′ end of SEQ ID NO:X. This primer preferably spansabout 100 nucleotides. This primer set is then used in a polymerasechain reaction under the following set of conditions : 30 seconds, 95°C.; 1 minute, 56° C.; 1 minute, 70° C. This cycle is repeated 32 timesfollowed by one 5 minute cycle at 70° C. Human, mouse, and hamster DNAis used as template in addition to a somatic cell hybrid panelcontaining individual chromosomes or chromosome fragments (Bios, Inc).The reactions is analyzed on either 8% polyacrylamide gels or 3.5%agarose gels. Chromosome mapping is determined by the presence of anapproximately 100 bp PCR fragment in the particular somatic cell hybrid.

Example 5 Bacterial Expression of a Polypeptide

[0405] A polynucleotide encoding a polypeptide of the present inventionis amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ ends of the DNA sequence, as outlined in Example 1, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Amp^(I)), a bacterial origin of replication(ori), an IPTG-regulatable promoter/operator (P/O), a ribosome bindingsite (RBS), a 6-histidine tag (6-His), and restriction enzyme cloningsites.

[0406] The pQE-9 vector is digested with BamHI and XbaI and theamplified fragment is ligated into the pQE-9 vector maintaining thereading frame initiated at the bacterial RBS. The ligation mixture isthen used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) whichcontains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kan^(I)). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

[0407] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(Isopropyl-B-D-thiogalacto pyranoside) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression.

[0408] Cells are grown for an extra 3 to 4 hours. Cells are thenharvested by centrifugation (20 mins at 6000Xg). The cell pellet issolubilized in the chaotropic agent 6 Molar Guanidine HCl by stirringfor 3-4 hours at 4° C. The cell debris is removed by centrifugation, andthe supernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni—NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni—NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

[0409] Briefly, the supernatant is loaded onto the column in 6 Mguanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 Mguanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

[0410] The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni—NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

[0411] In addition to the above expression vector, the present inventionfurther includes an expression vector comprising phage operator andpromoter elements operatively linked to a polynucleotide of the presentinvention, called pHE4a. (ATCC Accession Number 209645, deposited onFeb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferasegene as a selection marker, 2) an E. coli origin of replication, 3) a T5phage promoter sequence, 4) two lac operator sequences, 5) aShine-Delgarno sequence, and 6) the lactose operon repressor gene(lacIq). The origin of replication (oriC) is derived from pUC19 (LTI,Gaithersburg, Md.). The promoter sequence and operator sequences aremade synthetically.

[0412] DNA can be inserted into the pHEa by restricting the vector withNdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product ona gel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to the PCRprotocol described in Example 1, using PCR primers having restrictionsites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer).The PCR insert is gel purified and restricted with compatible enzymes.The insert and vector are ligated according to standard protocols.

[0413] The engineered vector could easily be substituted in the aboveprotocol to express protein in a bacterial system.

Example 6 Purification of a Polypeptide from an Inclusion Body

[0414] The following alternative method can be used to purify apolypeptide expressed in E coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10° C.

[0415] Upon completion of the production phase of the E. colifermentation, the cell culture is cooled to 4-10° C. and the cellsharvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech).On the basis of the expected yield of protein per unit weight of cellpaste and the amount of purified protein required, an appropriate amountof cell paste, by weight, is suspended in a buffer solution containing100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to ahomogeneous suspension using a high shear nixer.

[0416] The cells are then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000 ×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

[0417] The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000 ×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4° C. overnight to allow furtherGuHCl extraction.

[0418] Following high speed centrifugation (30,000 ×g) to removeinsoluble particles, the GuHCl solubilized protein is refolded byquickly mixing the GuHCl extract with 20 volumes of buffer containing 50mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. Therefolded diluted protein solution is kept at 4° C. without mixing for 12hours prior to further purification steps.

[0419] To clarify the refolded polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 μm membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 nm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

[0420] Fractions containing the polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the polypeptide (determined, for instance, by 16% SDS-PAGE)are then pooled.

[0421] The resultant polypeptide should exhibit greater than 95% purityafter the above refolding and purification steps. No major contaminantbands should be observed from Commassie blue stained 16% SDS-PAGE gelwhen 5 μg of purified protein is loaded. The purified protein can alsobe tested for endotoxin/LPS contamination, and typically the LPS contentis less than 0.1 ng/ml according to LAL assays.

Example 7 Cloning and Expression of a Polypeptide in a BaculovirusExpression System

[0422] In this example, the plasmid shuttle vector pA2 is used to inserta polynucleotide into a baculovirus to express a polypeptide. Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI, Xba I and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate a viable virus thatexpress the cloned polynucleotide.

[0423] Many other baculovirus vectors can be used in place of the vectorabove, such as pAc373, pVL941, and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

[0424] Specifically, the cDNA sequence contained in the deposited clone,including the AUG initiation codon and the naturally associated leadersequence identified in Table 1, is amplified using the PCR protocoldescribed in Example 1. If the naturally occurring signal sequence isused to produce the secreted protein, the pA2 vector does not need asecond signal peptide. Alternatively, the vector can be modified (pA2GP) to include a baculovirus leader sequence, using the standard methodsdescribed in Summers et al., “A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures,” Texas AgriculturalExperimental Station Bulletin No. 1555 (1987).

[0425] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

[0426] The plasmid is digested with the corresponding restrictionenzymes and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.).

[0427] The fragment and the dephosphorylated plasmid are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria containing the plasmid are identified by digesting DNAfrom individual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

[0428] Five μg of a plasmid containing the polynucleotide isco-transfected with 1.0 μg of a commercially available linearizedbaculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego,Calif.), using the lipofection method described by Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413-7417 (1987). One μg of BaculoGold™ virusDNA and 5 μg of the plasmid are mixed in a sterile well of a microtiterplate containing 50 μl of serum-free Grace's medium (Life TechnologiesInc., Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μlGrace's medium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27° C. The transfection solution is then removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. Cultivation is then continued at 27° C. for four days.

[0429] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10.) After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C.

[0430] To verify the expression of the polypeptide, Sf9 cells are grownin Grace's medium supplemented with 10% heat-inactivated FBS. The cellsare infected with the recombinant baculovirus containing thepolynucleotide at a multiplicity of infection (“MOI”) of about 2. Ifradiolabeled proteins are desired, 6 hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). After 42 hours,5 μCi of ³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham)are added. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoiadiography (if radiolabeled).

[0431] Microsequencing of the amino acid sequence of the amino terminusof purified protein may be used to determine the amino terminal sequenceof the produced protein.

Example 8 Expression of a Polypeptide in Mammalian Cells

[0432] The polypeptide of the present invention can be expressed in amammalian cell. A typical mammalian expression vector contains apromoter element, which mediates the initiation of transcription ofmRNA, a protein coding sequence, and signals required for thetermination of transcription and polyadenylation of the transcript.Additional elements include enhancers, Kozak sequences and interveningsequences flanked by donor and acceptor sites for RNA splicing. Highlyefficient transcription is achieved with the early and late promotersfrom SV40, the long terminal repeats (LTRs) from Retroviruses, e.g.,RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter).

[0433] Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0.Mammalian host cells that could be used include, human Hela, 293, H9 andJurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quailQC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[0434] Alternatively, the polypeptide can be expressed in stable celllines containing the polynucleotide integrated into a chromosome. Theco-transfection with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transfectedcells.

[0435] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful in developing cell lines that carry several hundred oreven several thousand copies of the gene of interest. (See, e.g., Alt,F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. andMa, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. andSydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selectionmarker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992).Using these markers, the mammalian cells are grown in selective mediumand the cells with the highest resistance are selected. These cell linescontain the amplified gene(s) integrated into a chromosome. Chinesehamster ovary (CHO) and NSO cells are often used for the production ofproteins.

[0436] Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146),the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No.209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985).) Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors also contain the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene,and the mouse DHFR gene under control of the SV40 early promoter.

[0437] Specifically, the plasmid pC6, for example, is digested withappropriate restriction enzymes and then dephosphorylated using calfintestinal phosphates by procedures known in the art. The vector is thenisolated from a 1% agarose gel.

[0438] A polynucleotide of the present invention is amplified accordingto the protocol outlined in Example 1. If the naturally occurring signalsequence is used to produce the secreted protein, the vector does notneed a second signal peptide. Alternatively, if the naturally occurringsignal sequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891.)

[0439] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

[0440] The amplified fragment is then digested with the same restrictionenzyme and purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL- 1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

[0441] Chinese hamster ovary cells lacking an active DHFR gene is usedfor transfection. Five μg of the expression plasmid pC6 is cotransfectedwith 0.5 μg of the plasrmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 9 Protein Fusions

[0442] The polypeptides of the present invention are preferably fused toother proteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example 5; see also EP A 394,827;Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion toIgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclearlocalization signals fused to the polypeptides of the present inventioncan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule, or the protocol described inExample 5.

[0443] Briefly, the human Fc portion of the IgG molecule can be PCRamplified, using primers that span the 5′ and 3′ ends of the sequencedescribed below. These primers also should have convenient restrictionenzyme sites that will facilitate cloning into an expression vector,preferably a mammalian expression vector.

[0444] For example, if pC4 (Accession No. 209646) is used, the human Fcportion can be ligated into the BamHI cloning site. Note that the 3′BamHI site should be destroyed. Next, the vector containing the human Fcportion is re-restricted with BamHI, linearizing the vector, and apolynucleotide of the present invention, isolated by the PCR protocoldescribed in Example 1, is ligated into this BamHI site. Note that thepolynucleotide is cloned without a stop codon, otherwise a fusionprotein will not be produced.

[0445] If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.) Human IgG Fc region:GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCC (SEQ ID NO:1)CAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 10 Production of an Antibody from a Polypeptide

[0446] The antibodies of the present invention can be prepared by avariety of methods. (See, Current Protocols, Chapter 2.) For example,cells expressing a polypeptide of the present invention is administeredto an animal to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of the secreted proteinis prepared and purified to render it substantially free of naturalcontaminants. Such a preparation is then introduced into an animal inorder to produce polyclonal antisera of greater specific activity.

[0447] In the most preferred method, the antibodies of the presentinvention are monoclonal antibodies (or protein binding fragmentsthereof). Such monoclonal antibodies can be prepared using hybridomatechnology. (Köhler et al., Nature 256:495 (1975); Köhler et al., Eur.J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures involveimmunizing an animal (preferably a mouse) with polypeptide or, morepreferably, with a secreted polypeptide-expressing cell. Such cells maybe cultured in any suitable tissue culture medium; however, it ispreferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

[0448] The splenocytes of such mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP20), available fromthe ATCC. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981).) Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding thepolypeptide.

[0449] Alternatively, additional antibodies capable of binding to thepolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens. and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the protein-specificantibody can be blocked by the polypeptide. Such antibodies compriseanti-idiotypic antibodies to the protein-specific antibody and can beused to immunize an animal to induce formation of furtherprotein-specific antibodies.

[0450] It will be appreciated that Fab and F(ab′)2 and other fragmentsof the antibodies of the present invention may be used according to themethods disclosed herein. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)2 fragments). Alternatively,secreted protein-binding fragments can be produced through theapplication of recombinant DNA technology or through syntheticchemistry.

[0451] For in vivo use of antibodies in humans, it may be preferable touse “humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494;, Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).)

Example 11 Production Of Secreted Protein For High-Throughput ScreeningAssays

[0452] The following protocol produces a supernatant containing apolypeptide to be tested. This supernatant can then be used in theScreening Assays described in Examples 13-20.

[0453] First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stocksolution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516FBiowhittaker) for a working solution of 50 ug/ml. Add 200 ul of thissolution to each well (24 well plates) and incubate at RT for 20minutes. Be sure to distribute the solution over each well (note: a12-channel pipetter may be used with tips on every other channel).Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS(Phosphate Buffered Saline). The PBS should remain in the well untiljust prior to plating the cells and plates may be poly-lysine coated inadvance for up to two weeks.

[0454] Plate 293T cells (do not carry cells past P+20) at 2×10⁵cells/well in 0.5 ml DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/Lglucose and L-glutamine (12-604F Biowhittaker))/10% heat inactivatedFBS(14-503F Biowhittaker)/1× Penstrep(17-602E Biowhittaker). Let thecells grow overnight.

[0455] The next day, mix together in a sterile solution basin: 300 ulLipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter,aliquot approximately 2 ug of an expression vector containing apolynucleotide insert, produced by the methods described in Examples 8or 9, into an appropriately labeled 96-well round bottom plate. With amulti-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixtureto each well. Pipette up and down gently to mix. Incubate at RT 15-45minutes. After about 20 minutes, use a multi-channel pipetter to add 150ul Optimem I to each well. As a control, one plate of vector DNA lackingan insert should be transfected with each set of transfections.

[0456] Preferably, the transfection should be performed by tag-teamingthe following tasks. By tag-teaming, hands on time is cut in half, andthe cells do not spend too much time on PBS. First, person A aspiratesoff the media from four 24-well plates of cells, and then person Brinses each well with 0.5-1 ml PBS. Person A then aspirates off PBSrinse, and person B, using a 12-channel pipetter with tips on everyother channel, adds the 200 ul of DNA/Lipofectamine/Optimem I complex tothe odd wells first, then to the even wells, to each row on the 24-wellplates. Incubate at 37° C. for 6 hours.

[0457] While cells are incubating, prepare appropriate media, either 1%BSA in DMEM with 1× penstrep, or CHO-5 media (116.6 mg/L of CaC12(anhyd); 0.00130 mg/L CuSO₄-5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417mg/L of FeSO₄-7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/Lof MgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L ofNaH₂PO₄-H₂O; 71.02 mglL of Na₂HPO4; 0.4320 mg/L of ZnSO₄-7H₂O; 0.002mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L ofDL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L ofLinolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid;0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L ofPluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551mg/L of D-Glucose; 130.85 mg/ml of L- Alanine; 147.50 mg/ml ofL-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂O; 6.65 mg/ml of L-AsparticAcid; 29.56 mg/ml of L-Cystine-2HCL-H₂O; 31.29 mg/ml of L-Cystine-2HCL;7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/mlof Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂O; 106.97 mg/ml ofL-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL;32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/mlof L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂O; 99.65mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-CaPantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid;15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L ofPyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin;3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and 0.680 mg/L ofVitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L ofSodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 2 uM of Ethanolamine;0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrincomplexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrincomplexed with Oleic Acid; and 10 mg/L of Methyl-B-Cyclodextrincomplexed with Retinal) with 2 mm glutamine and 1× penstrep. (BSA(81-068-3 Bayer) 100 gm dissolved in 1 L DMEM for a 10% BSA stocksolution). Filter the media and collect 50 ul for endotoxin assay in 15ml polystyrene conical.

[0458] The transfection reaction is terminated, preferably bytag-teaming, at the end of the incubation period. Person A aspirates offthe transfection media, while person B adds 1.5 ml appropriate media toeach well. Incubate at 37° C. for 45 or 72 hours depending on the mediaused: 1% BSA for 45 hours or CHO-5 for 72 hours.

[0459] On day four, using a 300 ul multichannel pipetter, aliquot 600 ulin one 1 ml deep well plate and the remaining supernatant into a 2 mldeep well. The supernatants from each well can then be used in theassays described in Examples 13-20.

[0460] It is specifically understood that when activity is obtained inany of the assays described below using a supernatant, the activityoriginates from either the polypeptide directly (e.g., as a secretedprotein) or by the polypeptide inducing expression of other proteins,which are then secreted into the supernatant. Thus, the inventionfurther provides a method of identifying the protein in the supernatantcharacterized by an activity in a particular assay.

Example 12 Construction of GAS Reporter Construct

[0461] One signal transduction pathway involved in the differentiationand proliferation of cells is called the Jaks-STATs pathway. Activatedproteins in the Jaks-STATs pathway bind to gamma activation site “GAS”elements or interferon-sensitive responsive element (“ISRE”), located inthe promoter of many genes. The binding of a protein to these elementsalter the expression of the associated gene.

[0462] GAS and ISRE elements are recognized by a class of transcriptionfactors called Signal Transducers and Activators of Transcription, or“STATs.” There are six members of the STATs family. Stat1 and Stat3 arepresent in many cell types, as is Stat2 (as response to IFN-alpha iswidespread). Stat4 is more restricted and is not in many cell typesthough it has been found in T helper class I, cells after treatment withIL-12. Stat5 was originally called mammary growth factor, but has beenfound at higher concentrations in other cells including myeloid cells.It can be activated in tissue culture cells by many cytokines.

[0463] The STATs are activated to translocate from the cytoplasm to thenucleus upon tyrosine phosphorylation by a set of kinases known as theJanus Kinase (“Jaks”) family. Jaks represent a distinct family ofsoluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. Thesekinases display significant sequence similarity and are generallycatalytically inactive in resting cells.

[0464] The Jaks are activated by a wide range of receptors summarized inthe Table below. (Adapted from review by Schidler and Darnell, Ann. Rev.Biochem. 64:621-51 (1995).) A cytokine receptor family, capable ofactivating Jaks, is divided into two groups: (a) Class 1 includesreceptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15,Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share aconserved cysteine motif (a set of four conserved cysteines and onetryptophan) and a WSXWS motif (a membrane proximal region encodingTrp-Ser-Xxx-Trp-Ser (SEQ ID NO:2)).

[0465] Thus, on binding of a ligand to a receptor, Jaks are activated,which in turn activate STATs, which then translocate and bind to GASelements. This entire process is encompassed in the Jaks-STATs signaltransduction pathway.

[0466] Therefore, activation of the Jaks-STATs pathway, reflected by thebinding of the GAS or the ISRE element, can be used to indicate proteinsinvolved in the proliferation and differentiation of cells. For example,growth factors and cytokines are known to activate the Jaks-STATspathway. (See Table below.) Thus, by using GAS elements linked toreporter molecules, activators of the Jaks-STATs pathway can beidentified. JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS (elements) or ISREIFN family IFN-a/B + + − − 1,2,3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 >IFP) Il-10 + ? ? − 1,3 gp130 family IL-6 (Pleiotrophic) + + + ? 1,3 GAS(IRF1 > Lys6 > IFP) Il-11 (Pleiotrophic) ? + ? ? 1,3 OnM (Pleiotrophic)? + + ? 1,3 LIF (Pleiotrophic) ? + + ? 1,3 CNTF (Pleiotrophic) −/+ + + ?1,3 G-CSF (Pleiotrophic) ? + ? ? 1,3 IL-12 (Pleiotrophic) + − + + 1,3g-C family IL-2 (lymphocytes) − + − + 1,3,5 GAS IL-4 (lymph/myeloid) − +− + 6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GAS IL-9(lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15 ? +? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >> Ly6)IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growthhormone family GH ? − + − 5 PRL ? +/− + − 1,3,5 EPO ? − + − 5 GAS(B-CAS > IRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1,3GAS (IRF1) PDGF ? + + − 1,3 CSF-1 ? + + − 1,3 GAS (not IRF1)

[0467] To construct a synthetic GAS containing promoter element, whichis used in the Biological Assays described in Examples 13-14, a PCRbased strategy is employed to generate a GAS-SV40 promoter sequence. The5′ primer contains four tandem copies of the GAS binding site found inthe IRF1 promoter and previously demonstrated to bind STATs uponinduction with a range of cytokines (Rothman et al., Immunity 1:457-468(1994).), although other GAS or ISRE elements can be used instead. The5′ primer also contains 18 bp of sequence complementary to the SV40early promoter sequence and is flanked with an XhoI site. The sequenceof the 5′ primer is:5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCG (SEQ ID NO:3)AAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′

[0468] The downstream primer is complementary to the SV40 promoter andis flanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQID NO:4)

[0469] PCR amplification is performed using the SV40 promoter templatepresent in the B-gal:promoter plasmid obtained from Clontech. Theresulting PCR fragment is digested with XhoI/Hind III and subcloned intoBLSK2-. (Stratagene.) Sequencing with forward and reverse primersconfirms that the insert contains the following sequence: 5′:+E,unsCTCGAGATTTCCCCGAAATCTAGA1TFTCCCCGAAATGATTTCCCCGAAATG (SEQ ID NO:5)ATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTT TGCAAA+E,unsAAGCTT:3′

[0470] With this GAS promoter element linked to the SV40 promoter, aGAS:SEAP2 reporter construct is next engineered. Here, the reportermolecule is a secreted alkaline phosphatase, or “SEAP.” Clearly,however, any reporter molecule can be instead of SEAP, in this or in anyof the other Examples. Well known reporter molecules that can be usedinstead of SEAP include chloramphenicol acetyltransferase (CAT),luciferase, alkaline phosphatase, B-galactosidase, green fluorescentprotein (GFP), or any protein detectable by an antibody.

[0471] The above sequence confirmed synthetic GAS-SV40 promoter elementis subcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

[0472] Thus, in order to generate mammalian stable cell lines expressingthe GAS-SEAP reporter, the GAS-SEAP cassette is removed from theGAS-SEAP vector using SalI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding as described in Examples 13-14.

[0473] Other constructs can be made using the above description andreplacing GAS with a different promoter sequence. For example,construction of reporter molecules containing NFK-B and EGR promotersequences are described in Examples 15 and 16. However, many otherpromoters can be substituted using the protocols described in theseExamples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can besubstituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB,II-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used totest reporter construct activity, such as HELA (epithelial), HUVEC(endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), orCardiomyocyte.

Example 13 High-Throughput Screening Assay for T-cell Activity.

[0474] The following protocol is used to assess T-cell activity byidentifying factors, such as growth factors and cytokines, that mayproliferate or differentiate T-cells. T-cell activity is assessed usingthe GAS/SEAP/Neo construct produced in Example 12. Thus, factors thatincrease SEAP activity indicate the ability to activate the Jaks-STATSsignal transduction pathway. The T-cell used in this assay is JurkatT-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCCAccession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582)cells can also be used.

[0475] Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In orderto generate stable cell lines, approximately 2 million Jurkat cells aretransfected with the GAS-SEAP/neo vector using DMRIE-C (LifeTechnologies)(transfection procedure described below). The transfectedcells are seeded to a density of approximately 20,000 cells per well andtransfectants resistant to 1 mg/ml genticin selected. Resistant coloniesare expanded and then tested for their response to increasingconcentrations of interferon gamma. The dose response of a selectedclone is demonstrated.

[0476] Specifically, the following protocol will yield sufficient cellsfor 75 wells containing 200 ul of cells. Thus, it is either scaled up,or performed in multiple to generate sufficient cells for multiple 96well plates. Jurkat cells are maintained in RPMI +10% serum with 1%Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug ofplasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul ofDMRIE-C and incubate at room temperature for 15-45 mins.

[0477] During the incubation period, count cell concentration, spin downthe required a number of cells (10⁷ per transfection), and resuspend inOPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37° C. for 6 hrs.After the incubation, add 10 ml of RPMI +15% serum.

[0478] The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treatedwith supernatants containing a polypeptide as produced by the protocoldescribed in Example 11.

[0479] On the day of treatment with the supernatant, the cells should bewashed and resuspended in fresh RPMI +10% serum to a density of 500,000cells per ml. The exact number of cells required will depend on thenumber of supernatants being screened. For one 96 well plate,approximately 10 million cells (for 10 plates, 100 million cells) arerequired.

[0480] Transfer the cells to a triangular reservoir boat, in order todispense the cells into a 96 well dish, using a 12 channel pipette.Using a 12 channel pipette, transfer 200 ul of cells into each well(therefore adding 100,000 cells per well).

[0481] After all the plates have been seeded, 50 ul of the supematantsare transferred directly from the 96 well plate containing thesupernatants into each well using a 12 channel pipette. In addition, adose of exogenous interferon gamma (0.1, 1.0, 10 ng) is added to wellsH9, H10, and H11 to serve as additional positive controls for the assay.

[0482] The 96 well dishes containing Jurkat cells treated withsupernatants are placed in an incubator for 48 hrs (note: this time isvariable between 48-72 hrs). 35 ul samples from each well are thentransferred to an opaque 96 well plate using a 12 channel pipette. Theopaque plates should be covered (using sellophene covers) and stored at−20° C. until SEAP assays are performed according to Example 17. Theplates containing the remaining treated cells are placed at 4° C. andserve as a source of material for repeating the assay on a specific wellif desired.

[0483] As a positive control, 100 Unit/ml interferon gamma can be usedwhich is known to activate Jurkat T cells. Over 30 fold induction istypically observed in the positive control wells.

[0484] The above protocol may be used in the generation of bothtransient, as well as, stable transfected cells, which would be apparentto those of skill in the art.

Example 14 High-Throughput Screening Assay Identifying Myeloid Activity

[0485] The following protocol is used to assess myeloid activity byidentifying factors, such, as growth factors and cytokines, that mayproliferate or differentiate myeloid cells. Myeloid cell activity isassessed using the GAS/SEAP/Neo construct produced in Example 12. Thus,factors that increase SEAP activity indicate the ability to activate theJaks-STATS signal transduction pathway. The myeloid cell used in thisassay is U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 canbe used.

[0486] To transiently transfect U937 cells with the GAS/SEAP/Neoconstruct produced in Example 12, a DEAE-Dextran method (Kharbanda et.al., 1994, Cell Growth & Differentiation, 5:259-265) is used. First,harvest 2×10e⁷ U937 cells and wash with PBS. The U937 cells are usuallygrown in RPMI 1640 medium containing 10% heat-inactivated fetal bovineserum (FBS) supplemented with 100 units/ml penicillin and 100 mg/mlstreptomycin.

[0487] Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffercontaining 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mMNaCl, 5 mM KCl, 375 uM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂.Incubate at 37° C. for 45 min.

[0488] Wash the cells with RPMI 1640 medium containing 10% FBS and thenresuspend in 10 ml complete medium and incubate at 37° C. for 36 hr.

[0489] The GAS-SEAP/U937 stable cells are obtained by growing the cellsin 400 ug/ml G418. The G418-free medium is used for routine growth butevery one to two months, the cells should be re-grown in 400 ug/ml G418for couple of passages.

[0490] These cells are tested by harvesting 1×10⁸ cells (this is enoughfor ten 96-well plates assay) and wash with PBS. Suspend the cells in200 ml above described growth medium, with a final density of 5×10⁵cells/ml. Plate 200 ul cells per well in the 96-well plate (or 1×10⁵cells/well).

[0491] Add 50 ul of the supernatant prepared by the protocol describedin Example 11. Incubate at 37° C. for 48 to 72 hr. As a positivecontrol, 100 Unit/ml interferon gamma can be used which is known toactivate U937 cells. Over 30 fold induction is typically observed in thepositive control wells. SEAP assay the supernatant according to theprotocol described in Example 17.

Example 15 High-Throughput Screening Assay Identifying Neuronal Activity

[0492] When cells undergo differentiation and proliferation, a group ofgenes are activated through many different signal transduction pathways.One of these genes, EGR1 (early growth response gene 1), is induced invarious tissues and cell types upon activation. The promoter of EGR1 isresponsible for such induction. Using the EGR1 promoter linked toreporter molecules, activation of cells can be assessed.

[0493] Particularly, the following protocol is used to assess neuronalactivity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells)are known to proliferate and/or differentiate by activation with anumber of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF(nerve growth factor), and EGF (epidermal growth factor). The EGR1 geneexpression is activated during this treatment. Thus, by stablytransfecting PC12 cells with a construct containing an EGR promoterlinked to SEAP reporter, activation of PC12 cells can be assessed.

[0494] The EGR/SEAP reporter construct can be assembled by the followingprotocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al.,Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNAusing the following primers: 5′GCGCTCGAGGGATGACAGCGATAGAA (SEQ ID NO:6)CCCCGG-3′ 5′GCGAAGCTTCGCGACTCCCCGGAT (SEQ ID NO:7) CCGCCTC-3′

[0495] Using the GAS:SEAP/Neo vector produced in Example 12, EGR1amplified product can then be inserted into this vector. Linearize theGAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing theGAS/SV40 stuffer. Restrict the EGR1 amplified product with these sameenzymes. Ligate the vector and the EGR1 promoter.

[0496] To prepare 96 well-plates for cell culture, two mls of a coatingsolution (1:30 dilution of collagen type I (Upstate Biotech Inc.Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cmplate or 50 ml per well of the 96-well plate, and allowed to air dry for2 hr.

[0497] PC12 cells are routinely grown in RPMI-1640 medium (BioWhittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. #12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplementedwith 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated10 cm tissue culture dish. One to four split is done every three to fourdays. Cells are removed from the plates by scraping and resuspended withpipetting up and down for more than 15 times.

[0498] Transfect the EGR/SEAP/Neo construct into PC12 using theLipofectamine protocol described in Example 11. EGR-SEAP/PC12 stablecells are obtained by growing the cells in 300 ug/ml G418. The G418-freemedium is used for routine growth but every one to two months, the cellsshould be re-grown in 300 ug/ml G418 for couple of passages.

[0499] To assay for neuronal activity, a 10 cm plate with cells around70 to 80% confluent is screened by removing the old medium. Wash thecells once with PBS (Phosphate buffered saline). Then starve the cellsin low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBSwith antibiotics) overnight.

[0500] The next morning, remove the medium and wash the cells with PBS.Scrape off the cells from the plate, suspend the cells well in 2 ml lowserum medium. Count the cell number and add more low serum medium toreach final cell density as 5×10⁵ cells/ml.

[0501] Add 200 ul of the cell suspension to each well of 96-well plate(equivalent to 1×10⁵ cells/well). Add 50 ul supernatant produced byExample 11, 37° C. for 48 to 72 hr. As a positive control, a growthfactor known to activate PC12 cells through EGR can be used, such as 50ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAPis typically seen in the positive control wells. SEAP assay thesupernatant according to Example 17.

Example 16 High-Throughput Screening Assay for T-cell Activity

[0502] NF-κB (Nuclear Factor κB) is a transcription factor activated bya wide variety of agents including the inflammatory cytokines IL-1 andTNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposureto LPS or thrombin, and by expression of certain viral gene products. Asa transcription factor, NF-κB regulates the expression of genes involvedin immune cell activation, control of apoptosis (NF-κB appears to shieldcells from apoptosis), B and T-cell development, anti-viral andantimicrobial responses, and multiple stress responses.

[0503] In non-stimulated conditions, NF-κB is retained in the cytoplasmwith I-κB (Inhibitor κB). However, upon stimulation, I-κB isphosphorylated and degraded, causing NF-κB to shuttle to the nucleus,thereby activating transcription of target genes. Target genes activatedby NF-κB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

[0504] Due to its central role and ability to respond to a range ofstimuli, reporter constructs utilizing the NF-κB promoter element areused to screen the supernatants produced in Example 11. Activators orinhibitors of NF-κB would be useful in treating diseases. For example,inhibitors of NF-κB could be used to treat those diseases related to theacute or chronic activation of NF-κB, such as rheumatoid arthritis.

[0505] To construct a vector containing the NF-κB promoter element, aPCR based strategy is employed. The upstream primer contains four tandemcopies of the NF-κB binding site (GGGGACTTTCCC) (SEQ ID NO:8), 18 bp ofsequence complementary to the 5′ end of the SV40 early promotersequence, and is flanked with an XhoI site:5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGAC (SEQ ID NO:9)TTTCCATCCTGCCATCTCAATTAG:3′

[0506] The downstream primer is complementary to the 3′ end of the SV40promoter and is flanked with a Hind III site:5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO:4)

[0507] PCR amplification is performed using the SV40 promoter templatepresent in the pB-gal:promoter plasmid obtained from Clontech. Theresulting PCR fragment is digested with XhoI and Hind III and subclonedinto BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirmsthe insert contains the following sequence:5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCC (SEQ ID NO:10)ATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT: 3′

[0508] Next, replace the SV40 minimal promoter element present in thepSEAP2-promoter plasmid (Clontech) with this NF-κB/SV40 fragment usingXhoI and HindIII. However, this vector does not contain a neomycinresistance gene, and therefore, is not preferred for mammalianexpression systems.

[0509] In order to generate stable mammalian cell lines, theNF-κB/SV40/SEAP cassette is removed from the above NF-κB/SEAP vectorusing restriction enzymes SalI and NotI, and inserted into a vectorcontaining neomycin resistance. Particularly, the NF-κB/SV40/SEAPcassette was inserted into pGFP-1 (Clontech), replacing the GFP gene,after restricting pGFP-1 with SalI and NotI.

[0510] Once NF-κB/SV40/SEAP/Neo vector is created, stable Jurkat T-cellsare created and maintained according to the protocol described inExample 13. Similarly, the method for assaying supernatants with thesestable Jurkat T-cells is also described in Example 13. As a positivecontrol, exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10,and H11, with a 5-10 fold activation typically observed.

Example 17 Assay for SEAP Activity

[0511] As a reporter molecule for the assays described in Examples13-16, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat.BP-400) according to the following general procedure. The TropixPhospho-light Kit supplies the Dilution, Assay, and Reaction Buffersused below.

[0512] Prime a dispenser with the 2.5× Dilution Buffer and dispense 15μl of 2.5× dilution buffer into Optiplates containing 35 μl of asupernatant. Seal the plates with a plastic sealer and incubate at 65°C. for 30 min. Separate the Optiplates to avoid uneven heating.

[0513] Cool the samples to room temperature for 15 minutes. Empty thedispenser and prime with the Assay Buffer. Add 50 μl Assay Buffer andincubate at room temperature 5 min. Empty the dispenser and prime withthe Reaction Buffer (see the table below). Add 50 μl Reaction Buffer andincubate at room temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on luminometer, one should treat 5 plates at each timeand start the second set 10 minutes later.

[0514] Read the relative light unit in the luminometer. Set H12 asblank, and print the results. An increase in chemiluminescence indicatesreporter activity. Reaction Buffer Formulation: # of plates Rxn bufferdiluent (ml) CSPD (ml) 10  60 3 11  65 3.25 12  70 3.5 13  75 3.75 14 80 4 15  85 4.25 16  90 4.5 17  95 4.75 18 100 5 19 105 5.25 20 110 5.521 115 5.75 22 120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 1457.25 28 150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.7534 180 9 35 185 9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40210 10.5 41 215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46240 12 47 245 12.25 48 250 12.5 49 255 12.75 50 260 13

Example 18 High-Throughput Screening Assay Identifying Changes in SmallMolecule Concentration and Membrane Permeability

[0515] Binding of a ligand to a receptor is known to alter intracellularlevels of small molcules, such as calcium, potassium, sodium, and pH, aswell as alter membrane potential. These alterations can be measured inan assay to identify supernatants which bind to receptors of aparticular cell. Although the following protocol describes an assay forcalcium, this protocol can easily be modified to detect changes inpotassium, sodium, pH, membrane potential, or any other small moleculewhich is detectable by a fluorescent probe.

[0516] The following assay uses Fluorometric Imaging Plate Reader(“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes)that bind small molecules. Clearly, any fluorescent molecule detecting asmall molecule can be used instead of the calcium fluorescent molecule,fluo-4 (Molecular Probes, Inc. catalog no. F-14202), used here.

[0517] For adherent cells, seed the cells at 10,000-20,000 cells/well ina Co-star black 96-well plate with clear bottom. The plate is incubatedin a CO₂ incubator for 20 hours. The adherent cells are washed two timesin Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

[0518] A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acidDMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is addedto each well. The plate is incubated at 37° C. in a CO₂ incubator for 60min. The plate is washed four times in the Biotek washer with HBSSleaving 100 ul of buffer.

[0519] For non-adherent cells, the cells are spun down from culturemedia. Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-mlconical tube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSOis added to each ml of cell suspension. The tube is then placed in a 37°C. water bath for 30-60 min. The cells are washed twice with HBSS,resuspended to 1×10⁶ cells/ml, and dispensed into a microplate, 100ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate isthen washed once in Denley CellWash with 200 ul, followed by anaspiration step to 100 ul final volume.

[0520] For a non-cell based assay, each well contains a fluorescentmolecule, such as fluo-4. The supernatant is added to the well, and achange in fluorescence is detected.

[0521] To measure the fluorescence of intracellular calcium, the FLIPRis set for the following parameters: (1) System gain is 300-800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul.Increased emission at 530 nm indicates an extracellular signaling eventwhich has resulted in an increase in the intracellular Ca⁺⁺concentration.

Example 19 High-Throughput Screening Assay Identifying Tyrosine KinaseActivity

[0522] The Protein Tyrosine Kinases (PTK) represent a diverse group oftransmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies. In addition there are a large family ofRPTKs for which the corresponding ligand is unknown. Ligands for RPTKsinclude mainly secreted small proteins, but also membrane-bound andextracellular matrix proteins.

[0523] Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

[0524] Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, the identification of novel human secretedproteins capable of activating tyrosine kinase signal transductionpathways are of interest. Therefore, the following protocol is designedto identify those novel human secreted proteins capable of activatingthe tyrosine kinase signal transduction pathways.

[0525] Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalge Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type I collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at4° C. Cell growth on these plates is assayed by seeding 5,000 cells/wellin growth medium and indirect quantitation of cell number through use ofalamarBlue as described by the manufacturer Alamar Biosciences, Inc.(Sacramento, Calif.) after 48 hr. Falcon plate covers #3071 from BectonDickinson (Bedford, Mass.) are used to cover the Loprodyne Silent ScreenPlates. Falcon Microtest III cell culture plates can also be used insome proliferation experiments.

[0526] To prepare extracts, A431 cells are seeded onto the nylonmembranes of Loprodyne plates (20,000/200ml/well) and cultured overnightin complete medium. Cells are quiesced by incubation in serum-free basalmedium for 24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50ul of the supernatant produced in Example 11, the medium was removed and100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% TritonX-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P207 and a cocktail of proteaseinhibitors (# 1836170) obtained from Boeheringer Mannheim (Indianapolis,Ind.) is added to each well and the plate is shaken on a rotating shakerfor 5 minutes at 4° C. The plate is then placed in a vacuum transfermanifold and the extract filtered through the 0.45 mm membrane bottomsof each well using house vacuum. Extracts are collected in a 96-wellcatch/assay plate in the bottom of the vacuum manifold and immediatelyplaced on ice. To obtain extracts clarified by centrifugation, thecontent of each well, after detergent solubilization for 5 minutes, isremoved and centrifuged for 15 minutes at 4° C. at 16,000× g.

[0527] Test the filtered extracts for levels of tyrosine kinaseactivity. Although many methods of detecting tyrosine kinase activityare known, one method is described here.

[0528] Generally, the tyrosine kinase activity of a supernatant isevaluated by determining its ability to phosphorylate a tyrosine residueon a specific substrate (a biotinylated peptide). Biotinylated peptidesthat can be used for this purpose include PSK1 (corresponding to aminoacids 6-20 of the cell division kinase cdc2-p34) and PSK2 (correspondingto amino acids 1-17 of gastrin). Both peptides are substrates for arange of tyrosine kinases and are available from Boehringer Mannheim.

[0529] The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 ul of 5× Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of SodiumVanadate(1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30° C. for 2 min. Initial the reactionby adding 10 ul of the control enzyme or the filtered supernatant.

[0530] The tyrosine kinase assay reaction is then terminated by adding10 ul of 120 mm EDTA and place the reactions on ice.

[0531] Tyrosine kinase activity is determined by transferring 50 ulaliquot of reaction mixture to a microtiter plate (MTP) module andincubating at 37° C. for 20 min. This allows the streptavadin coated 96well plate to associate with the biotinylated peptide. Wash the MTPmodule with 300 ul/well of PBS four times. Next add 75 ul ofanti-phospotyrosine antibody conjugated to horse radishperoxidase(anti-P-Tyr-POD(0.5 u/ml)) to each well and incubate at 37° C.for one hour. Wash the well as above.

[0532] Next add 100 ul of peroxidase substrate solution (BoehringerMannheim) and incubate at room temperature for at least 5 mins (up to 30min). Measure the absorbance of the sample at 405 nm by using ELISAreader. The level of bound peroxidase activity is quantitated using anELISA reader and reflects the level of tyrosine kinase activity.

Example 20 High-Throughput Screening Assay Identifying PhosphorylationActivity

[0533] As a potential alternative and/or compliment to the assay ofprotein tyrosine kinase activity described in Example 19, an assay whichdetects activation (phosphorylation) of major intracellular signaltransduction intermediates can also be used. For example, as describedbelow one particular assay can detect tyrosine phosphorylation of theErk-1 and Erk-2 kinases. However, phosphorylation of other molecules,such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src,Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as anyother phosphoserine, phosphotyrosine, or phosphothreonine molecule, canbe detected by substituting these molecules for Erk-1 or Erk-2 in thefollowing assay.

[0534] Specifically, assay plates are made by coating the wells of a96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at roomtemp, (RT). The plates are then rinsed with PBS and blocked with 3%BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2commercial monoclonal antibodies (100 ng/well) against Erk-1 and Erk-2(1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules,this step can easily be modified by substituting a monoclonal antibodydetecting any of the above described molecules.) After 3-5 rinses withPBS, the plates are stored at 4° C. until use.

[0535] A431 cells are seeded at 20,000/well in a 96-well Loprodynefilterplate and cultured overnight in growth medium. The cells are thenstarved for 48 hr in basal medium (DMEM) and then treated with EGF (6ng/well) or 50 ul of the supernatants obtained in Example 11 for 5-20minutes. The cells are then solubilized and extracts filtered directlyinto the assay plate.

[0536] After incubation with the extract for 1 hr at RT, the wells areagain rinsed. As a positive control, a commercial preparation of MAPkinase (10 ng/well) is used in place of A431 extract. Plates are thentreated with a commercial polyclonal (rabbit) antibody (1 ug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Wallac DELFIA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation.

Example 21 Method of Determining Alterations in a Gene Corresponding toa Polynucleotide

[0537] RNA isolated from entire families or individual patientspresenting with a phenotype of interest (such as a disease) is beisolated. cDNA is then generated from these RNA samples using protocolsknown in the art. (See, Sambrook.) The cDNA is then used as a templatefor PCR, employing primers surrounding regions of interest in SEQ IDNO:X. Suggested PCR conditions consist of 35 cycles at 95° C. for 30seconds; 60-120 seconds at 52-58° C.; and 60-120 seconds at 70° C.,using buffer solutions described in Sidransky, D., et al., Science252:706 (1991).

[0538] PCR products are then sequenced using primers labeled at their 5′end with T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons isalso determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations is then cloned andsequenced to validate the results of the direct sequencing.

[0539] PCR products is cloned into T-tailed vectors as described inHolton, T. A. and Graham, M. W., Nucleic Acids Research, 19:1156 (1991)and sequenced with T7 polymerase (United States Biochemical). Affectedindividuals are identified by mutations not present in unaffectedindividuals.

[0540] Genomic rearrangements are also observed as a method ofdetermining alterations in a gene corresponding to a polynucleotide.Genomic clones isolated according to Example 2 are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson, Cg. et al., Methods Cell Biol.35:73-99 (1991). Hybridization with the labeled probe is carried outusing a vast excess of human cot-1 DNA for specific hybridization to thecorresponding genomic locus.

[0541] Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 22 Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

[0542] A polypeptide of the present invention can be detected in abiological sample, and if an increased or decreased level of thepolypeptide is detected, this polypeptide is a marker for a particularphenotype. Methods of detection are numerous, and thus, it is understoodthat one skilled in the art can modify the following assay to fit theirparticular needs.

[0543] For example, antibody-sandwich ELISAs are used to detectpolypeptides in a sample, preferably a biological sample. Wells of amicrotiter plate are coated with specific antibodies, at a finalconcentration of 0.2 to 10 ug/ml. The antibodies are either monoclonalor polyclonal and are produced by the method described in Example 10.The wells are blocked so that non-specific binding of the polypeptide tothe well is reduced.

[0544] The coated wells are then incubated for >2 hours at RT with asample containing the polypeptide. Preferably, serial dilutions of thesample should be used to validate results. The plates are then washedthree times with deionized or distilled water to remove unboundedpolypeptide.

[0545] Next, 50 ul of specific antibody-alkaline phosphatase conjugate,at a concentration of 25-400 ng, is added and incubated for 2 hours atroom temperature. The plates are again washed three times with deionizedor distilled water to remove unbounded conjugate.

[0546] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) orp-nitrophenyl phosphate (NPP) substrate solution to each well andincubate 1 hour at room temperature. Measure the reaction by armicrotiter plate reader. Prepare a standard curve, using serialdilutions of a control sample, and plot polypeptide concentration on theX-axis (log scale) and fluorescence or absorbance of the Y-axis (linearscale). Interpolate the concentration of the polypeptide in the sampleusing the standard curve.

Example 23 Formulating a Polypeptide

[0547] The secreted polypeptide composition will be formulated and dosedin a fashion consistent with good medical practice, taking into accountthe clinical condition of the individual patient (especially the sideeffects of treatment with the secreted polypeptide alone), the site ofdelivery, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The “effectiveamount” for purposes herein is thus determined by such considerations.

[0548] As a general proposition, the total pharmaceutically effectiveamount of secreted polypeptide administered parenterally per dose willbe in the range of about 1 μg/kg/day to 10 mg/kg/day of patient bodyweight, although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the secreted polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

[0549] Pharmaceutical compositions containing the secreted protein ofthe invention are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[0550] The secreted polypeptide is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. etal., Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl methacrylate)(R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R.Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langeret al.) or poly-D- (−)-3-hydroxybutyric acid (EP 133,988).Sustained-release compositions also include liposomally entrappedpolypeptides. Liposomes containing the secreted polypeptide are preparedby methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad.Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal secreted polypeptide therapy.

[0551] For parenteral administration, in one embodiment, the secretedpolypeptide is formulated generally by mixing it at the desired degreeof purity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically acceptable carrier, i.e., one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation. For example,the formulation preferably does not include oxidizing agents and othercompounds that are known to be deleterious to polypeptides.

[0552] Generally, the formulations are prepared by contacting thepolypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

[0553] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0554] The secreted polypeptide is typically formulated in such vehiclesat a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10mg/ml, at a pH of about 3 to 8. It will be understood that the use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of polypeptide salts.

[0555] Any polypeptide to be used for therapeutic administration can besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticpolypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

[0556] Polypeptides ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized polypeptide using bacteriostaticWater-for-Injection.

[0557] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides of the present invention may be employed inconjunction with other therapeutic compounds.

Example 24 Method of Treating Decreased Levels of the Polypeptide

[0558] It will be appreciated that conditions caused by a decrease inthe standard or normal expression level of a secreted protein in anindividual can be treated by administering the polypeptide of thepresent invention, preferably in the secreted form. Thus, the inventionalso provides a method of treatment of an individual in need of anincreased level of the polypeptide comprising administering to such anindividual a pharmaceutical composition comprising an amount of thepolypeptide to increase the activity level of the polypeptide in such anindividual.

[0559] For example, a patient with decreased levels of a polypeptidereceives a daily dose 0.1 -100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in the secreted form.The exact details of the dosing scheme, based on administration andformulation, are provided in Example 23.

Example 25 Method of Treating Increased Levels of the Polypeptide

[0560] Antisense technology is used to inhibit production of apolypeptide of the present invention. This technology is one example ofa method of decreasing levels of a polypeptide, preferably a secretedform, due to a variety of etiologies, such as cancer.

[0561] For example, a patient diagnosed with abnormally increased levelsof a polypeptide is administered intravenously antisense polynucleotidesat 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment isrepeated after a 7-day rest period if the treatment was well tolerated.The formulation of the antisense polynucleotide is provided in Example23.

Example 26 Method of Treatment Using Gene Therapy

[0562] One method of gene therapy transplants fibroblasts, which arecapable of expressing a polypeptide, onto a patient. Generally,fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. The flasks are then incubated at 37° C. forapproximately one week.

[0563] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

[0564] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0565] The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 1. Preferably, the 5′primer contains an EcoRI site and the 3′ primer includes a HindIII site.Equal quantities of the Moloney murine sarcoma virus linear backbone andthe amplified EcoRI and HindIII fragment are added together, in thepresence of T4 DNA ligase. The resulting mixture is maintained underconditions appropriate for ligation of the two fragments. The ligationmixture is then used to transform bacteria HB101, which are then platedonto agar containing kanamycin for the purpose of confirming that thevector has the gene of interest properly inserted.

[0566] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0567] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether protein is produced.

[0568] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

Example 27 Method of Treatment Using Gene Therapy—In Vivo

[0569] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) sequences into an animal to increase ordecrease the expression of the polypeptide. The polynucleotide of thepresent invention may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the polypeptide by thetarget tissue. Such gene therapy and delivery techniques and methods areknown in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat.Nos. 5,693,622, 5,705,151, 5,580,859; Tabata H. et al. (1997)Cardiovasc. Res. 35(3):470-479, Chao J et al. (1997) Pharmacol. Res.35(6):517-522, Wolff J. A. (1997) Neuromuscul. Disord. 7(5):314-318,Schwartz B. et al. (1996) Gene Ther. 3(5):405-411, Tsurumi Y. et al.(1996) Circulation 94(12):3281-3290 (incorporated herein by reference).

[0570] The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

[0571] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the polynucleotides of the present invention may alsobe delivered in liposome formulations (such as those taught in FelgnerP. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. etal. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods wellknown to those skilled in the art.

[0572] The polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapies techniques,one major advantage of introducing naked nucleic acid sequences intotarget cells is the transitory nature of the polynucleotide synthesis inthe cells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

[0573] The polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

[0574] For the naked polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 g/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

[0575] The dose response effects of injected polynucleotide in muscle invivo is determined as follows. Suitable template DNA for production ofmRNA coding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

[0576] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The template DNA is injected in 0.1 ml of carrierin a 1 cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

[0577] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15um cross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in huinans andother animals using naked DNA.

Example 28 Transgenic Animals

[0578] The polypeptides of the invention can also be expressed intransgenic animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats,sheep, cows and non-human primates, e.g., baboons, monkeys, andchimpanzees may be used to generate transgenic animals. In a specificembodiment, techniques described herein or otherwise known in the art,are used to express polypeptides of the invention in humans, as part ofa gene therapy protocol.

[0579] Any technique known in the art may be used to introduce thetransgene (i.e., polynucleotides of the invention) into animals toproduce the founder lines of transgenic animals. Such techniquesinclude, but are not limited to, pronuclear microinjection (Paterson etal., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al.,Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191(1989)); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)),blastocysts or embryos; gene targeting in embryonic stem cells (Thompsonet al., Cell 56:313-321 (1989)); electroporation of cells or embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of thepolynucleotides of the invention using a gene gun (see, e.g., Ulmer etal., Science 259:1745 (1993); introducing nucleic acid constructs intoembryonic pleuripotent stem cells and transferring the stem cells backinto the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,Cell 57:717-723 (1989); etc. For a review of such techniques, seeGordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989),which is incorporated by reference herein in its entirety.

[0580] Any technique known in the art may be used to produce transgenicclones containing polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813, (1997)).

[0581] The present invention provides for transgenic animals that carrythe transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

[0582] Once transgenic animals have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

[0583] Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

[0584] Transgenic animals of the invention have uses which include, butare not limited to, animal model systems useful in elaborating thebiological function of polypeptides of the present invention, studyingconditions and/or disorders associated with aberrant expression, and inscreening for compounds effective in ameliorating such conditions and/ordisorders.

Example 29: Knock-Out Animals

[0585] Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

[0586] In further embodiments of the invention, cells that aregenetically engineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

[0587] Alternatively, the cells can be incorporated into a matrix andimplanted in the body, e.g., genetically engineered fibroblasts can beimplanted as part of a skin graft; genetically engineered endothelialcells can be implanted as part of a lymphatic or vascular graft. (See,for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan &Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated byreference herein in its entirety).

[0588] When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

[0589] Transgenic and “knock-out” animals of the invention have useswhich include, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying conditions and/or disorders associated with aberrantexpression, and in screening for compounds effective in amelioratingsuch conditions and/or disorders.

[0590] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0591] The entire disclosure of each document cited (including patents,patent applications, journal articles, abstracts, laboratory manuals,books, or other disclosures) in the Background of the Invention,Detailed Description, and Examples is hereby incorporated herein byreference. Further, the hard copy of the sequence listing submittedherewith and the corresponding computer readable form are bothincorporated herein by reference in their entireties.

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95% identical to asequence selected from the group consisting of: (a) a polynucleotidefragment of SEQ ID NO:X or a polynucleotide fragment of the cDNAsequence included in ATCC Deposit No:Z, which is hybridizable to SEQ IDNO:X; (b) a polynucleotide encoding a polypeptide fragment of SEQ IDNO:Y or a polypeptide fragment encoded by the cDNA sequence included inATCC Deposit No:Z, which is hybridizable to SEQ ID NO:X; (c) apolynucleotide encoding a polypeptide domain of SEQ ID NO:Y or apolypeptide domain encoded by the cDNA sequence included in ATCC DepositNo:Z, which is hybridizable to SEQ ID NO:X; (d) a polynucleotideencoding a polypeptide epitope of SEQ ID NO:Y or a polypeptide epitopeencoded by the cDNA sequence included in ATCC Deposit No:Z, which ishybridizable to SEQ ID NO:X; (e) a polynucleotide encoding a polypeptideof SEQ ID NO:Y or the cDNA sequence included in ATCC Deposit No:Z, whichis hybridizable to SEQ ID NO:X, having biological activity; (f) apolynucleotide which is a variant of SEQ ID NO:X; (g) a polynucleotidewhich is an allelic variant of SEQ ID NO:X; (h) a polynucleotide whichencodes a species homologue of the SEQ ID NO:Y; (i) a polynucleotidecapable of hybridizing under stringent conditions to any one of thepolynucleotides specified in (a)-(h), wherein said polynucleotide doesnot hybridize under stringent conditions to a nucleic acid moleculehaving a nucleotide sequence of only A residues or of only T residues.2. The isolated nucleic acid molecule of claim 1 , wherein thepolynucleotide fragment comprises a nucleotide sequence encoding asecreted protein.
 3. The isolated nucleic acid molecule of claim 1 ,wherein the polynucleotide fragment comprises a nucleotide sequenceencoding the sequence identified as SEQ ID NO:Y or the polypeptideencoded by the cDNA sequence included in ATCC Deposit No:Z, which ishybridizable to SEQ ID NO:X.
 4. The isolated nucleic acid molecule ofclaim 1 , wherein the polynucleotide fragment comprises the entirenucleotide sequence of SEQ ID NO:X or the cDNA sequence included in ATCCDeposit No:Z, which is hybridizable to SEQ ID NO:X.
 5. The isolatednucleic acid molecule of claim 2 , wherein the nucleotide sequencecomprises sequential nucleotide deletions from either the C-terminus orthe N-terminus.
 6. The isolated nucleic acid molecule of claim 3 ,wherein the nucleotide sequence comprises sequential nucleotidedeletions from either the C-terminus or the N-terminus.
 7. A recombinantvector comprising the isolated nucleic acid molecule of claim 1 .
 8. Amethod of making a recombinant host cell comprising the isolated nucleicacid molecule of claim 1 .
 9. A recombinant host cell produced by themethod of claim 8 .
 10. The recombinant host cell of claim 9 comprisingvector sequences.
 11. An isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence selected from the groupconsisting of: (a) a polypeptide fragment of SEQ ID NO:Y or the encodedsequence included in ATCC Deposit No:Z; (b) a polypeptide fragment ofSEQ ID NO:Y or the encoded sequence included in ATCC Deposit No:Z,having biological activity; (c) a polypeptide domain of SEQ ID NO:Y orthe encoded sequence included in ATCC Deposit No:Z; (d) a polypeptideepitope of SEQ ID NO:Y or the encoded sequence included in ATCC DepositNo:Z; (e) a secreted form of SEQ ID NO:Y or the encoded sequenceincluded in ATCC Deposit No:Z; (f) a full length protein of SEQ ID NO:Yor the encoded sequence included in ATCC Deposit No:Z; (g) a variant ofSEQ ID NO:Y; (h) an allelic variant of SEQ ID NO:Y; or (i) a specieshomologue of the SEQ ID NO:Y.
 12. The isolated polypeptide of claim 11 ,wherein the secreted form or the full length protein comprisessequential amino acid deletions from either the C-terminus or theN-terminus.
 13. An isolated antibody that binds specifically to theisolated polypeptide of claim 11 .
 14. A recombinant host cell thatexpresses the isolated polypeptide of claim 11 .
 15. A method of makingan isolated polypeptide comprising: (a) culturing the recombinant hostcell of claim 14 under conditions such that said polypeptide isexpressed; and (b) recovering said polypeptide.
 16. The polypeptideproduced by claim 15 .
 17. A method for preventing, treating, orameliorating a medical condition, comprising administering to amammalian subject a therapeutically effective amount of the polypeptideof claim 11 or the polynucleotide of claim 1 .
 18. A method ofdiagnosing a pathological condition or a susceptibility to apathological condition in a subject comprising: (a) determining thepresence or absence of a mutation in the polynucleotide of claim 1 ; and(b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.
 19. A method of diagnosing a pathological condition or asusceptibility to a pathological condition in a subject comprising: (a)determining the presence or amount of expression of the polypeptide ofclaim 11 in a biological sample; and (b) diagnosing a pathologicalcondition or a susceptibility to a pathological condition based on thepresence or amount of expression of the polypeptide.
 20. A method foridentifying a binding partner to the polypeptide of claim 11 comprising:(a) contacting the polypeptide of claim 11 with a binding partner; and(b) determining whether the binding partner effects an activity of thepolypeptide.
 21. The gene corresponding to the cDNA sequence of SEQ IDNO:Y.
 22. A method of identifying an activity in a biological assay,wherein the method comprises: (a) expressing SEQ ID NO:X in a cell; (b)isolating the supernatant; (c) detecting an activity in a biologicalassay; and (d) identifying the protein in the supernatant having theactivity.
 23. The product produced by the method of claim 20 .